Genus Tigriopus Research Articles

Prezygotic reproductive barriers in precopulatory behavior of tidepool copepod species.

Complexity in prezygotic mating behavior can contribute to the emergence of sexual incompatibility and reproductive isolation. In this study, we performed behavioral tests with two tidepool copepod species of the genus Tigriopus to explore the possibility of precopulatory behavioral isolation. We found that interspecific mating attempts failed prior to genital contact, and that this failure occurred at different behavioral steps between reciprocal pairings. Our results suggest that prezygotic barriers may exist at multiple points of the behavioral process on both male and female sides, possibly due to interspecific differences in mate-recognition cues used at those "checkpoints". While many copepod species are known to show unique precopulatory mate-guarding behavior, the potential contribution of prezygotic behavioral factors to their isolation is not widely recognized. The pattern of sequential mate-guarding behaviors may have allowed diversification of precopulatory communication and contributed to the evolutionary diversity of the Tigriopus copepods.

Phylogenetic relationships among species of Tigriopus (Multicrustacea: Copepoda: Harpacticoida) with comments on its biogeography

Here we present some hypothetical phylogenetic relationships and the evolutionary history of the harpacticoid copepod genus Tigriopus Norman, 1869 using morphological data. Cladistic analyses were performed with 21 morphological characters, including 15 ingroup and eight outgroup species. Inferred topology from Bayesian inference supported the monophyletic status of the genus, and revealed two main evolutionary lineages. One of these lineages (the brachydactylus-iagi lineage) comprises species from the Indo-Pacific, the northern Pacific, the southern Pacific, and the southern Atlantic; it is supported by the tetrasetose female P5 endopod. The fulvus-angulatus lineage is composed of three clusters with species from the north Atlantic and the Mediterranean Sea (the fulvus-brevicornis clade), the northwestern Pacific and the Indo-Pacific (the japonicus-sirindhoranae clade), and from the southern Pacific, the Indian Ocean, and the Southern Ocean (the crozettensis-angulatus clade). The groundpattern of the mandibular basis of the fulvus-angulatus lineage seems to include a bisetose mandibular basis, and, the presence of three setae on this segment in the japonicus-sirindhoranae clade is interpreted here as a character reversal. Biogeographic analyses suggest that the tropical Indo-Pacific region is the most probable ancestral area of the genus that diversified through vicariance events.

Two new species of Tigriopus Norman, 1869 from Chonburi Province, Thailand (Crustacea: Copepoda: Harpacticidae)

Two new species of the family Harpacticidae Dana, 1846, Tigriopus namsaiensis sp. nov. and T. wannapaensis sp. nov. were found in algal washings at Namsai and Wannapa beaches in Chonburi Province, Thailand. There are currently 14 known species in the genus, two of which, T. thailandensis Chullasorn, Ivanenko, Dahms, Kangtia Yang, 2012 and T. sirindhornae Chullasorn, Dahms Klangsin, 2013 were also discovered in Thailand. The genus Tigriopus Norman, 1869 has a worldwide distribution with nine species recorded from the Pacific Ocean (seven in the North Pacific and two in the South Pacific), four species from the Atlantic and adjacent seas, two species from the South Indian Ocean, and one species from Antarctica. Sexual dimorphism is expressed in the antennule, antenna, P2, P5, P6, and segmentation of the urosome. Tigriopus namsaiensis sp. nov. and T. wannapaensis sp. nov. are closely related to T. thailandensis and T. sirindhornae in sharing the following characters: fewer sensilla on the prosome; antennary exopod with one seta on exp-2; P4 exp-3 with two inner setae; female P5 baseoendopod with five setae; male P2 enp-2 with a seta fused to the segment; and male P5 exopod with five setae. Tigriopus namsaiensis sp. nov. closely resembles T. thailandensis, but the new species is characterized by four setae on the maxillulary coxa (three setae in T. thailandensis), the outermost seta on the female P6 is plumose (instead of the middle one), the lateral seta on the apical segment of the antennary exopod is discrete at the base (instead of fused to the segment), and the outermost seta of the male P5 exopod is plumose (instead of spinulose). Tigriopus wannapaensis sp. nov. is very closely related to T. sirindhornae, but the new species differs from the latter by the following characteristics: 2-segmented mandibular exopod (3-segmented in T. sirindhornae); five setae on the maxillulary coxa (instead of three); all setae on female P6 are plumose (instead of two plumose and one pinnate); the lateral seta on the apical segment of the antennary exopod is fused to the segment (instead of discrete at the base); and the outermost seta on the male P5 exopod is plumose (instead of spinulose). A dichotomous identification key to the 16 valid species of Tigriopus is provided.

Range‐wide phylogeography and taxonomy of the marine rock pools dwellerTigriopus fulvus(Fischer, 1860) (Copepoda, Harpacticoida)

In the light of the wide distribution and ecological importance of the genus Tigriopus in coastal rock pool habitats, and of its frequent use in aquaculture and as a model organism, we investigated the identity of the Atlantic–Mediterranean Tigriopus populations and elucidated their taxonomy and patterns of morphological and genetic diversity. In order to reach these goals, an “integrative taxonomy” multisource approach was implemented. First, we investigated the constancy and taxonomical value of the morphological characters currently used to distinguish among the Tigriopus species occurring in the Mediterranean and in Eastern Atlantic area north of the Tropic of Cancer, and checked the actual morphological differences possibly present among topotypical samples of Tigriopus fulvus fulvus (Fischer, 1860) and its two subspecies Tigriopus fulvus adriaticus Van Douwe, 1913 and Tigriopus fulvus algiricus Monard, 1935. Then, we sequenced fragments of mitochondrial (12S) and nuclear (28S) genes. In the frame of this study, different “DNA taxonomy” approaches were implemented in order to check whether the subspecies of Tigriopus fulvus were actually lineages evolving independently, that is, valid species according to the “evolutionary genetic species concept.” The results coherently indicate the presence of a single species, characterized by constant morphology and a noteworthy geographically based genetic structure in the whole study area. No morphological or genetic support was found for the taxa of allegedly subspecific rank within T. fulvus, which are thus to be considered junior synonyms of T. fulvus s.s. Finally, a restricted locus typicus is established for T. fulvus, and a neotype is designated.

An improved genome assembly and annotation of the Antarctic copepod Tigriopus kingsejongensis and comparison of fatty acid metabolism between T. kingsejongensis and the temperate copepod T. japonicus

Copepods in the genus Tigriopus are widely distributed in the intertidal zone worldwide. To assess differences in fatty acid (FA) metabolism among congeneric species in this genus inhabiting polar and temperate environments, we analyzed and compared FA profiles of the Antarctic copepod Tigriopus kingsejongensis and the temperate copepod T. japonicus. Higher amounts of total FAs were found in the Antarctic copepod T. kingsejongensis than the temperate copepod T. japonicus under administration of the identical amount of Tetraselmis suecica. To determine the genomic basis for this, we identified fatty acid metabolism-related genes in an improved genome of T. kingsejongensis. The total length of the assembled genome was approximately 338 Mb with N50 = 1.473 Mb, 938 scaffolds, and a complete Benchmarking Universal Single-Copy Orthologs value of 95.8%. A total of 25,470 genes were annotated using newly established pipeline. We identified eight elongation of very long-chain fatty acid protein (Elovl) genes and nine fatty acid desaturase (Fad) genes in the genome of T. kingsejongensis. In addition, fatty acid profiling suggested that the duplicated Δ5/6 desaturase gene in T. kingsejongensis is likely to play an essential role in synthesis of different FAs in T. kingsejongensis to those in T. japonicus. However, further experimental research is required to validate our in silico findings. This study provides a better understanding of fatty acid metabolism in the Antarctic copepod T. kingsejongensis.

An account on the taxonomy and molecular diversity of a marine rock-pool dweller, Tigriopus fulvus (Copepoda, Harpacticoida)

The copepod genus Tigriopus Norman, 1869 is distributed worldwide in coastal rock pools and it is currently considered to include 14 valid species. Tigriopus fulvus (Fischer 1860), with its subspecies Tigriopus fulvus adriaticus Van Douwe 1913 and Tigriopus fulvus algiricus Monard 1935, and Tigriopus minutus Bozic 1960 are currently reported to occur in the Mediterranean area, but the actual diversity of the genus is currently unknown. We aimed to assess the actual identity of Mediterranean Tigriopus populations and to elucidate their taxonomy and pattern of genetic diversity. In order to reach these goals, a fragment of a mitochondrial DNA gene (cytochrome c oxidase subunit I, COI) was sequenced to be used as a reference marker. Our data suggest the presence of a single species characterized by a noteworthy geographically based genetic structure in the whole study area. The observed diversity pattern is tentatively ascribed here to a strong monopolization of the rock pools by the first immigrants that reached them. However, such a monopolization is periodically disrupted by local extinction events, which are frequent in the intrinsically unstable rock pool habitats. We propose the name “clockwork monopolization” for this pattern.

Complete mitochondrial genome of the Antarctic copepod Tigriopus kingsejongenesis (Harpacticoida, Harpacticidae)

The complete mitochondrial genome was sequenced from the Antarctic harpacticoid copepod Tigriopus kingsejongensis. The sequenced genome size was 14,940 bp, possessing different gene order and contents to those of the congeneric species T. japonicus and T. californicus in the genus Tigriopus. The mitochondrial genome of T. kingsejongensis has 13 protein-coding genes (PCGs), 2 rRNAs, and 22 tRNAs. Of the 13 PCGs, CO1, ND3, ATP6, and CO3 genes had incomplete stop codons T–, T–, TA–, and T–, respectively. Furthermore, the stop codons of the remaining 11 PCGs were TAA or TAG, while the start codon of 13 PCGs was ATG (CO1, ND4, Cytb, ND2, ND1, CO2, ND6, ATP6, CO3 genes), ATT (ND5, ATP8 genes), and ATA (ND3, ND4L genes), respectively. The nucleotide of 13 PCGs of T. kingsejongensis mitogenome showed 53.42, 52.17, and 49.10% similarities to the copepods T. japonicus, T. californicus, and Amphiascoides atopus, respectively, while the amino acid similarities were 64.38, 63.86, and 62.40%, respectively.

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior.

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Individual Culturing of <em>Tigriopus</em> Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Long-term laboratory culture causes contrasting shifts in tolerance to two marine pollutants in copepods of the genus Tigriopus.

Organismal chemical tolerance is often used to assess ecological risk and monitor water quality, yet tolerance can differ between field- and lab-raised organisms. In this study, we examined how tolerance to copper (Cu) and tributyltin oxide (TBTO) in two species of marine copepods, Tigriopus japonicus and T. californicus, changed across generations under benign laboratory culture (in the absence of pre-exposure to chemicals). Both copepod species exhibited similar chemical-specific changes in tolerance, with laboratory maintenance resulting in increased Cu tolerance and decreased TBTO tolerance. To assess potential factors underlying these patterns, chemical tolerance was measured in conjunction with candidate environmental variables (temperature, UV radiation, diet type, and starvation). The largest chemical-specific effect was found for starvation, which decreased TBTO tolerance but had no effect on Cu tolerance. Understanding how chemical-specific tolerance can change in the laboratory will be critical in strengthening bioassays and their applications for environmental protection and chemical management.

Sex-specific rejection in mate-guarding pair formation in the intertidal copepod, Tigriopus californicus.

Securing a potential mate is one of the most important processes in sexual reproduction of animals. Intertidal copepods of the genus Tigriopus show mate-guarding behavior where a male captures a female and continues to clasp her for up to two weeks prior to copulation. Although these copepods form a mate-guarding pair between a male and a female with high accuracy, interactions between the sexes in pair formation have not been well described and the mechanism allowing successful male-female pair formation is not yet understood. In this study, we performed experiments with Tigriopus californicus to analyze the behavior of both a capturer (male) and a captured individual (female or male) in formation of a guarding pair. While capturer males were attracted by both females and males, capture of virgin males was terminated in a significantly shorter time than that of virgin females. However, when presented freshly killed females or males, regardless of the sex of the body, capturer males continued to clasp the body for a comparable time as in an attempt on a living female. Our results suggest that a sex-specific rejection signal actively sent by captured males prevents male-male pair formation. Experiments also suggest that mated females reject an attempt of pair formation. To our knowledge, this is the first study to suggest involvement of active rejection by a captured individual in facilitation of reproductively successful male-female guarding pair formation in the genus Tigriopus.

Identification of the molecular components of a Tigriopus californicus (Crustacea, Copepoda) circadian clock

Copepods of the genus Tigriopus have been proposed as marine models for investigations of environmental perturbation. One rapidly increasing anthropogenic stressor for intertidal organisms is light pollution. Given the sensitivity of circadian rhythms to exogenous light, the genes/proteins of a Tigriopus circadian pacemaker represent a potential system for investigating the influences of artificial light sources on circadian behavior in an intertidal species. Here, the molecular components of a putative Tigriopus californicus circadian clock were identified using publicly accessible transcriptome data; the recently deduced circadian proteins of the copepod Calanus finmarchicus were used as a reference. Transcripts encoding homologs of all commonly recognized ancestral arthropod core clock proteins were identified (i.e. CLOCK, CRYPTOCHROME 2, CYCLE, PERIOD and TIMELESS), as were ones encoding proteins likely to modulate the core clock (i.e. CASEIN KINASE II, CLOCKWORK ORANGE, DOUBLETIME, PROTEIN PHOSPHATASE 1, PROTEIN PHOSPHATASE 2A, SHAGGY, SUPERNUMERARY LIMBS and VRILLE) or to act as inputs to it (i.e. CRYPTOCHROME 1). PAR DOMAIN PROTEIN 1 was the only circadian-associated protein not identified in Tigriopus; it appears absent in Calanus too. These data represent just the third full set of molecular components for a crustacean circadian pacemaker (Daphnia pulex and C. finmarchicus previously), and only the second obtained from transcribed sequences (C. finmarchicus previously). Given Tigriopus' proposed status as a model for investigating the influences of anthropogenic stressors in the marine environment, these data provide the first suite of gene/protein targets for understanding how light pollution may influence circadian physiology and behavior in an intertidal organism.

A new species of Tigriopus (Copepoda: Harpacticoida: Harpacticidae) from Thailand with a key to the species of the genus

A new species of the genus Tigriopus Norman, 1869 is described from Thailand. Tigriopus sirindhornae sp. nov. resembles other species in having cyclopiform shape, nine-segmented A1 in female, A2 allobasis with three-segmented exp, male lacking an abexopodal seta, male P2 enp II with distinct outer spiniform apophysis, both baseoendopod and exp of female P5 with five setae. The new species can be distinguished by having fewer sensillae; two-segmented mandibular exp; Mx1 praecoxa more spinulose, Mx2 with spinules on three syncoxal endites, and Mxp with spinules on outer margin. Additional characters related to the female P5 and P1 and the male A1 and P2 are also helpful to distinguish this species from its congeners. An identification key for the 13 known species of Tigriopus is also provided.http://www.zoobank.org/urn:lsid:zoobank.org:pub:ABA4C14F-E484-4883-8191-F7ECECC7A3D3

Phylogeography of the copepod Tigriopus japonicus along the Northwest Pacific rim

Copepod genus Tigriopus is distributed worldwide in coastal splash-pools, and is recognized as a suitable taxon for testing evolutionary and environmental hypotheses that are also relevant to water column research. Here, we present the complete DNA sequence (7709 bp) of a single ribosomal DNA (rDNA) unit of a Tigriopus species distributed along the coastlines of the Northwest Pacific ocean. The genomic organization of the Tigriopus japonicus (T japonicus) rDNA was observed to be 18S (1817 bp)-ITS1 (465 bp)-5.8S (165 bp)-ITS2 (226 bp)-28S (3628 bp)-IGS (1417 bp). The relationship of populations was studied using rDNA ITS (internal transcribed spacer) sequences from different geographical areas. The phylogenetic analysis of 18S rDNAs showed a single monophyletic clade of the Copepoda with an unresolved polytomy of the Harpacticoida, Monstrilloida and Siphonostomatoida. In Harpacticoida phytogeny, three species of Tigriopus were monophyletic; T. japonicus and Tigriopus californicus as sister species formed a cluster with Tigriopus brevicornis. Furthermore, phylogeographic analysis of ITS-5.8S sequences showed that Tigriopus from 15 Asian locations formed four distinct clades according to their geographical origins. When ITS-5.8S of those Asian specimens was compared with the corresponding mitochondrial COI (cytochrome c oxidase subunit I) gene data, a high similarity was found in the identical genotypes. However, they were different between the localities. This suggests that four groups identified from Asian Tigriopus would be different (sub)species, and the pattern of Asian Tigriopus genetic relatedness is due to their geographical separation.

Cannibalistic behaviour of rock-pool copepods: An experimental approach for space, food and kinship

Studies on cannibalism in harpacticoid copepods are restricted to predation on naupliar larvae in rock-pool harpacticoids of the genus Tigriopus. An earlier experimental study on the Mediterranean copepod Tigriopus fulvus indicated that females recognized their own larvae and preferentially preyed on nauplii other than their own. In a series of laboratory experiments, we tested if there were differences in naupliar predation as a function of crowding, food level and sex in Tigriopus brevicornis and T. fulvus. Results show that cannibalism was restricted to the first larval stages (N1 and N2). Both food availability and adult density significantly affected the predation rate. Contrary to earlier suggestions, adult males also preyed on the nauplii. We found no evidence that adults spare their own offspring, for neither T. fulvus nor T. brevicornis.This is in accordance with what one would expect for species having the life history characteristics of Tigriopus, i.e.: multiple broods and large number of offspring. Earlier results indicating parental care in Tigriopus must be taken with caution.

The complete mitochondrial genome of the intertidal copepod Tigriopus sp. (Copepoda, Harpactidae) from Korea and phylogenetic considerations

This paper reports on the basic characteristics of the mitochondrial genome of the intertidal copepod Tigriopus sp. from Korea, including its structural organization, base composition of rRNAs and protein-encoding genes, and the secondary structure of tRNAs. We amplified the complete mitochondrial DNA of the intertidal copepod Tigriopus sp. from Korea (sampling site Busan) by long-polymerase chain reaction (long-PCR) with conserved primers and sequenced this mitogenome by primer walking using flanking sequences as sequencing primers. The primer informations were obtained as expressed sequence tags (ESTs) from Tigriopus sp. The resultant Tigriopus sp. mitochondrial DNA sequence was 14,301 bp with a conserved structural organization, compared to that of T. japonicus from Japan with significant differences in several protein-coding regions including rRNAs, although the genomic organization of the mitochondrial genome was identical. In order to investigate biogeographic differences within the genus Tigriopus, we analyzed the CO1 gene by sequencing. This way, we compared several Tigriopus species from Korea, Japan, Hong Kong and Taiwan as well as other related species such as T. californicus, T. brevicornis and T. fulvus. The results further support the notion that the copepods display significantly different genomes within the same genus. These findings provide valuable genomic information for further studies on the population genetics and speciation processes within the genus Tigriopus.

Purification and sequencing of a mate-recognition protein from the copepod Tigriopus japonicus

Male copepods in the genus Tigriopus use surface proteins of female copepods to recognize appropriate mates. In a previous study of pre-copulatory mate-guarding in T. japonicus Mori, a monoclonal antibody was developed against one protein that contributed to the attractiveness of female T. japonicus as mate-guarding partners. In the present study, which was performed between December 1998 and November 2000, the antibody was used to purify two proteins from homogenates of pooled T. japonicus. A 70 kD protein was partially sequenced using mass spectrometry, revealing sequence similarity to α2-macroglobulin. It was shown that the 70 kD protein was highly expressed in stage CV females, the most attractive stage for mate-guarding. A purified 36 kD protein was also shown to be a cognate of α2-macroglobulin. Because the structure of both the 70 and 36 kD proteins suggested that protease activity and its inhibition might play a role in male mate choice, three protease inhibitors were tested in bioassays of male discrimination of conspecifics. The male's ability to discriminate among females was not completely blocked by the protease inhibitor, but their behavior was affected in more subtle ways. This suggests that the 70 kD protein, designated CSP70, is a variant of α2-macroglobulin, but with more specific activity.

Metapopulation dynamics of Tigriopus brevicornis (Harpacticoida) in intertidal rock pools

Rock pools above the level of mean high tide are frequently occupied by harpacticoid copepods of the genus Tigriopus. The network of populations in separate pools can be viewed as a metapopulation. However, in a dynamic environment subject to rainfall, tidal flushing and wave wash, there may be no particular structure to rock pool populations. Time series samples from pools at different levels on the shore were used to test for the presence of separate local population dynam- ics of T. brevicornis (Muller). Sample counts taken from the same pool 2 d apart showed autocorrela- tion in pools from the upper and middle of the range occupied by T. brevicornis. However, the dynamics of different pools were not strongly correlated. For counts of copepodites and adults, the degree of coupling between pools was a function of distance. The height of high tide affected counts in pools. Copepodite and adult numbers fell in pools at the lower end of the species range during spring tides. In comparison, there were spring-tide-associated increases in copepodite and adult numbers in pools above the mean height of high tide. Persistence of populations on the shore may therefore be aided by upshore movement of copepodites and adults during spring tides. Multivariate analysis of stage structure in pools suggested that pools acted as individual systems over a period of 31 d. The results indicate a degree of local population dynamics in separate pools, but the inferred metapopulation structure does not follow the classical model, since extinction events were rare in pools above the mean high tide level.

Karyological comparison among different Mediterranean populations of the genusTigriopus(Copepoda Harpacticoida)

Abstract The investigation carried out on three Mediterranean population of the genus Tigriopus showed that individuals from the populations Livorno and Rijeka are quite similar from the karyological point of view; on the contrary, the chromosome morphology of the population coming from Trapani is completely different. These results support the hypothesis that the North Tyrrhenian and the North Adriatic strains could represent the extremities of a chain of populations belonging to the same species, Tigriopus fulvus, whereas the South Tyrrhenian population could belong to a different species not yet recorded in this area. Further data collected on the North European species, Tigriopus brevicornis, indicate a great karyological difference between this species and the three Mediterranean populations studied. These results confirm the classification which assigns the populations found respectively in the North European and in the Mediterranean areas to different species.