RESUMEN
The shell of Artemia resting egg, which is a delicate multilayered envelope surrounding the inside diapause embryo, plays an important role in the survival strategy of Artemia. To date, the ultrastructure of resting eggshell has been studied for only handful populations, and knowledge about the diversity of shell structure is still limited. In this paper, resting eggs from 13 Artemia populations were studied by transmission electron microscopy. Results show that the basic configuration of resting eggshell is quite conservative, but variations are not uncommon in the fine ultrastructure of each main layer of the shell (e.g., the shape and distribution of the radially oriented pores in the cortical layer; the size, number and arrangement of chambers in the alveolar layer; and the development state of outer cuticular membrane [OCM]). The ultrastructural variation of eggshell seems not to be linked with species and reproductive mode of Artemia. Resting eggs from very high habitats (4300+ m above sea level [a.s.l.]) on Qinghai-Tibet Plateau and certain tropical salterns have a hypoplastic OCM, which may be related to the adaptation to habitat conditions such as low oxygen concentration. RESEARCH HIGHLIGHTS: Comparative study on resting eggs from 13 Artemia populations reveals high diversity in the fine structure of eggshell. Resting eggs from very high (4300+ m a.s.l.) habitats commonly have a hypoplastic OCM.
Asunto(s)
Artemia , Reproducción , Animales , Artemia/ultraestructura , Microscopía Electrónica de TransmisiónRESUMEN
The Qinghai-Tibet Plateau is one of the areas the richest in salt lakes and Artemia sites. As a result of climate warming and wetting, the areas of salt lakes on the plateau have been increasing, and the salinities have decreased considerably since 1990s. However, the impact of salinity change on the genetic diversity of Artemia is still unknown. Kyêbxang Co is the highest (4620 m above sea level) salt lake currently with commercial harvesting of Artemia resting eggs in the world, and harbors the largest Artemia population on the plateau. Its salinity had dropped from â¼67 ppt in 1998 to â¼39 ppt in 2019. Using 13 microsatellite markers and the mitochondrial cytochrome oxidase submit I (COI) gene, we analyzed the temporal changes of genetic diversity, effective population size and genetic structure of this Artemia population based on samples collected in 1998, 2007 and 2019. Our results revealed a steady decline of genetic diversity and significant genetic differentiation among the sampling years, which may be a consequence of genetic drift and the selection of decreased salinity. A decline of effective population size was also detected, which may be relative to the fluctuation in census population size, skewed sex ratio, and selection of the declined salinity. In 2007 and 2019, the Artemia population showed an excess of heterozygosity and significant deviation from Hardy-Weinberg Equilibrium (p < 0.001), which may be associated with the heterozygote advantage under low salinity. To comprehensively understand the impact of climate warming and wetting on Artemia populations on the plateau, further investigation with broad and intensive sampling are needed.
Asunto(s)
Artemia , Lagos , Humanos , Animales , Tibet , Lagos/química , Artemia/genética , Anostraca , Cambio Climático , Salinidad , Altitud , Variación GenéticaRESUMEN
Bird-mediated dispersal of resting eggs is the main mechanism for Artemia dispersal among catchments. The bisexual populations of Artemia urmiana species complex, which is here considered to be a collection of Artemia genetically close to the so-called "Western Asian Lineage", are mostly distributed in central and western Asia (i.e., in regions falling into the Central Asian Flyway of migratory birds) and live in diversified habitats. Little is known about the genetic relationships among these populations. Aiming to understand the population genetic characteristics and the roles of migratory birds on the dispersal and gene flow of this Artemia group, we evaluated the genetic diversity, genetic differentiation, and gene flow among 14 populations, with their altitudes ranging from 540 to 4870 m above sea level, using 13 microsatellite markers. Almost all populations exhibited high genetic diversity and heterozygote excess, which may be a consequence of combined effects of dispersal and hybridization. The global genetic differentiation (FST) value was 0.092, the pairwise FST values were 0.003-0.246. Discriminant analysis of principal components identified three genetic clusters, consisting of Urmia Lake (Iran), Zhundong (Xinjiang, China), and 12 Qinghai-Tibet Plateau populations, respectively. The among-population genetic differentiation seems to be a consequence of isolation by distance and adaptation to diversified habitats induced by altitudinal gradient. Historical gene flows are asymmetrical, and show an evolutionary source-sink dynamics, with Jingyu Lake (Xinjiang, China) population being the major source. These results support our hypothesis that in Qinghai-Tibet Plateau and surrounding areas the bird-mediated dispersal of Artemia may be biased towards from north to south and/or from higher altitude to lower altitude.
Asunto(s)
Anostraca , Artemia , Animales , Artemia/genética , Flujo Génico , Genética de Población , China , Aves , Variación GenéticaRESUMEN
Background: Artemia sinica is a brine shrimp species distributed in hypersaline salt lakes in northern China and Siberia and a successful invasive species in some coastal salterns. Although it is a commercially harvested and cultured species, knowledge of its reproductive characteristics is limited, and existing studies are often contradictory. The combined effects of temperature, salinity, and photoperiod on reproduction characteristics are experimentally studied to better understand its reproductive features. Methods: There were 36 combinations of three environmental factors (3 × 3 × 4), each with three or four levels, namely temperature (16, 25, 30 °C), photoperiod (6 L:18 D, 12 L:12D, 18 L:6D), and salinity (50, 100, 150, 200 PSU). In each treatment, 48 to 80 pairs of A. sinica from Yuncheng Salt Lake (Shanxi, China) were cultured. Females were observed daily for reproductive mode and the number of offspring produced. Results: Temperature, photoperiod, salinity, and their interactions significantly affected the lifespan and reproduction of A. sinica. The reproductive period was the longest and accounted for the largest proportion of life span at moderate temperature (25 °C). Total offspring, offspring per brood, and offspring per day increased as salinity decreased, and the number of broods per female was highest at 25 °C. Temperature, photoperiod, and salinity significantly influenced reproductive modes, and interactions among these factors were identified. Artemia sinica primarily reproduces oviparously under low temperature and short daylight conditions, and ovoviviparously under high temperature and long daylight conditions, with the maximum oviparity ratio recorded in treatments of 16 °C, 6L:18D, and 50 or 100 PSU. The maximum ovoviviparity ratio was recorded under 30 °C, 12L:12D, and 100 PSU. Unlike that documented for other Artemia species or populations, the brood size of A. sinica kept increasing throughout the reproductive period. It did not decline even in the last two broods. For the same brood number, the sizes of oviparous and ovoviviparous broods were similar. The length of the oviparous interval was often greater than that of the ovoviviparous interval, suggesting that oviparous offspring might require additional energy and time to construct the multi-layered eggshell. Compared to other species and populations, the A. sinica from Yuncheng Salt Lake has a relatively shorter pre-reproductive development time, a preference for ovoviviparity, and relatively higher fecundity and population growth capacity, making it a suitable culture species for obtaining fresh biomass.
Asunto(s)
Anostraca , Artemia , Animales , Femenino , Temperatura , Fotoperiodo , Salinidad , ReproducciónRESUMEN
The genus Parahubrechtia Gibson and Sundberg, 1999 was first described within the family Hubrechtiidae (class Pilidiophora) and subsequently transferred to the family Callineridae (class Palaeonemertea). Here we describe two new species, Parahubrechtia rayi sp. nov. from the Sea of Japan (Russia) and P. peri sp. nov. from the South China Sea (China). A phylogenetic analysis based on partial sequences of five nuclear and mitochondrial gene regions, 18S rRNA, 28S rRNA, histone H3, 16S rRNA, and COI, has confirmed the monophyly of the genus Parahubrechtia, and indicated a close relationship to Callinera Bergendal, 1900, whose monophyly is not confirmed. Both genera belong to the family Tubulanidae, with its junior synonym being Callineridae. Three major subclades are distinguished within the Tubulanidae: subclade Tubulanus s. str., subclade Tubulanus punctatus, and subclade Parahubrechtia + Callinera. The further status of Parahubrechtia depends on whether the paraphyly of Callinera is confirmed or not and how the problem of paraphyly of the genus Tubulanus Renier, 1804 is resolved.
RESUMEN
The transposon silencer piwi genes play important roles in germline determination and maintenance, gametogenesis, and stem-cell self-renewal, and the expression of certain piwi genes is indispensable for regeneration. Knowledge about piwi genes is needed for phylum Nemertea, which contains members (e.g., Lineus sanguineus) with formidable regeneration capacity. By searching the L. sanguineus genome, we identified six Argonaute genes including three ago (Ls-Ago2, Ls-Ago2a, and Ls-Ago2b) and three piwi (Ls-piwi1, Ls-piwi2, and Ls-piwi3) genes. In situ hybridization revealed that, in intact females, Ls-piwi2 and Ls-piwi3 were not expressed, while Ls-piwi1 was expressed in ovaries. During regeneration, Ls-piwi1 and Ls-pcna (proliferating cell nuclear antigen) had strong and similar expressions. The expression of Ls-piwi1 became indetectable while Ls-pcna continued to be expressed when the differentiation of new organs was finished. During anterior regeneration, expression signals of Ls-piwi2 and Ls-piwi3 were weak and only detected in the blastema stage. During posterior regeneration, no expression was observed for Ls-piwi2. To date, no direct evidence has been found for the existence of congenital stem cells in adult L. sanguineus. The "pluripotent cells" in regenerating tissues are likely to be dedifferentiated from other type(s) of cells.
Asunto(s)
Proteínas Argonautas/biosíntesis , Regulación de la Expresión Génica , Proteínas del Helminto/biosíntesis , Helmintos/metabolismo , Regeneración , Animales , Proteínas Argonautas/genética , Proteínas del Helminto/genética , Helmintos/genéticaRESUMEN
The genus Artemia Leach, 1819 is a cosmopolitan halophilic crustacean, consisting of bisexual species and obligate parthenogenetic populations. Asia is rich in Artemia biodiversity. More than 530 Artemia sites have been recorded from this area and more than 20 species/subspecies/variety names have been used for them. There exist various problems in the nomenclature, identification, and phylogenetic status of Artemia native to Asia, which are discussed in this paper.
RESUMEN
Urmia Lake, the largest natural habitat of the brine shrimp Artemia urmiana, has progressively desiccated over the last two decades, resulting in a loss of 80% of its surface area and producing thousands of hectares of arid salty land. This ecological crisis has seriously affected the lake's native biodiversity. Artemia urmiana has lost more than 90% of its population during the decade from 1994 (rainy period) to 2004 (drought period) due to salinity increasing to saturation levels (â¼300 g/l). We studied the influence of this ecological crisis on the genetic diversity of A. urmiana in Urmia Lake, based on one cyst collections in 1994 and 2004. AMOVA analysis on ISSR data demonstrated a 21% genetic variation and there was a 5.5% reduction of polymorphic loci between samples. PCoA showed that 77.42% and 68.75% of specimens clustered separately in 1994 and 2004, respectively. Our analyses of four marker genes revealed different genetic diversity patterns with a decrease of diversity at ITS1 and an increase for Na+/K+ ATPase. There was no notable difference in genetic variation detected for COI and 16S genes between the two periods. However, they represented distinctly different haplotypes. ITS1 and COI followed a population expansion model, whereas Na+/K+ ATPase and 16S were under demographic equilibrium without selective pressure in the 1994 samples. Neutrality tests confirmed the excess of rare historical and recent mutations present in COI and ITS1 in both samples. It is evident that a short-term ecological disturbance has impacted the genetic diversity and structure of A. urmiana.
RESUMEN
Outbreaks of ribbon worms observed in 2013, 2015, and 2017-2019 in the Han River Estuary, South Korea, have caused damage to local glass-eel fisheries. The Han River ribbon worms have been identified as Yininemertespratensis (Sun & Lu, 1998) based on not only morphological characteristics compared with the holotype and paratype specimens, but also DNA sequence comparison with topotypes freshly collected near the Yangtze River mouth, China. Using sequences of six gene markers (18S rRNA, 28S rRNA, histone H3, histone H4, 16S rRNA, and COI), the phylogenetic position of Y.pratensis was inferred among other heteronemerteans based on their sequences obtained from public databases. This analysis firmly placed Y.pratensis as a close relative to Apatronemertesalbimaculosa Wilfert & Gibson, 1974, which has been reported from aquarium tanks containing tropical freshwater plants in various parts of the world as well as a wild environment in Panama.
RESUMEN
A new species of parasitic copepod, Octopicola huanghaiensis n. sp., collected from the octopuses Amphioctopus fangsiao (d'Orbigny) and Octopus minor (Sasaki) (Octopoda: Octopodidae) in the Yellow Sea (off Qingdao, Shandong Province, China), is described. The new species is most similar to O. superba Humes, 1957, but can be distinguished from the latter by: (i) the third antennal segment having a different ornamentation; (ii) the fourth antennal segment of females much shorter than that in O. superba (49 vs 94 µm); (iii) males much smaller than females (mean body length 1.3 vs 2.0 mm, respectively) (vs similar male and female body size in O. superba, 1.9 mm and 1.8 mm respectively); and (iv) the presence of a spike at the posterior tip of each labrum flap. Octopicola huanghaiensis n. sp. is the first species of Octopicola Humes, 1957 reported from A. fangsiao and O. minor and is the only species of the family Octopicolidae Humes & Boxshall, 1996 known in North Pacific waters.
Asunto(s)
Copépodos/clasificación , Octopodiformes/parasitología , Animales , China , Copépodos/anatomía & histología , Femenino , Masculino , Océanos y Mares , Especificidad de la EspecieRESUMEN
Parthenogenetic Artemia from seven Chinese locations with different elevations and various ploidies are characterized by phenotypic and morphometric analyses. Our findings show that the studied populations exhibit dissimilar patterns of ovisac. Four phenotypic patterns of furca are qualified and one of them is shared among di-, tetra- and pentaploid Artemia. Results of discriminant analysis based on morphometric data reveal that tetra- and pentaploid populations are grouped together, but the Aqqikkol Lake population is clearly differentiated. Previous hypothesis/conclusion that polyploid Artemia are larger than diploids is only partly supported by the present results, which show that pentaploid and tetraploid populations are larger than the mostly diploid populations in terms of the total length, but the body size of the Aibi Lake triploids has not significant difference with the sympatric diploids and the mostly diploid Aqqikkol population that inhabit in very high altitude has the largest body size among all parthenogenetic populations. The founding confirms that body size of Artemia is following with Bergmann's rule.
Asunto(s)
Artemia/anatomía & histología , Artemia/genética , Ploidias , Estructuras Animales/anatomía & histología , Estructuras Animales/crecimiento & desarrollo , Animales , Artemia/clasificación , Artemia/crecimiento & desarrollo , Tamaño Corporal , China , Tamaño de los ÓrganosRESUMEN
The complete mitochondrial genome (mitogenome) of Tetrastemma olgarum is sequenced. It is 14,580 bp in length and contains 37 genes typical for metazoan mitogenomes. The gene order is identical to that of the previously published Hoplonemertea mitogenomes. All genes are encoded on the heavy strand except for trnT and trnP. The coding strand is AT-rich, accounting for 69.2% of overall nucleotide composition.
Asunto(s)
Genes de Helminto/fisiología , Genes Mitocondriales/fisiología , Genoma Mitocondrial/fisiología , Helmintos/genética , Animales , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
Heteronemerteans, such as Lineus ruber, L. viridis, Ramphogordius sanguineus, R. lacteus, Riseriellus occultus, and Micrura varicolor, share many similar external characters. Although several internal characters useful for distinguishing these nemertean species have been documented, their identification is based mostly on coloration, the shape of the head, and how they contract, which may not be always reliable. We sequenced the mitochondrial COI gene for 160 specimens recently collected from 27 locations around the world (provisionally identified as the above species, according to external characters and contraction patterns, with most of them as R. sanguineus). Based on these specimens, together with sequences of 16 specimens from GenBank, we conducted a DNA-based species delimitation/identification by means of statistical parsimony and phylogenetic analyses. Our results show that the analyzed specimens may contain nine species, which can be separated by large genetic gaps; heteronemerteans with an external appearance similar to R. sanguineus/Lineus ruber/L. viridis have high species diversity in European waters from where eight species can be discriminated. Our 42 individuals from Vancouver Island (Canada) are revealed to be R. sanguineus, which supports an earlier argument that nemerteans reported as L. ruber or L. viridis from the Pacific Northwest may refer to this species. We report R. sanguineus from Chile, southern China, and the species is also distributed on the Atlantic coast of South America (Argentina). In addition, present analyses reveal the occurrence of L. viridis in Qingdao, which is the first record of the species from Chinese waters.
Asunto(s)
Distribución Animal , Variación Genética , Invertebrados/genética , Animales , Invertebrados/clasificación , Filogenia , Especificidad de la EspecieRESUMEN
Of the 14 nominal species that are now or have ever been assigned to the genus Paranemertes Coe, 1901 , four have been reported to have stylets with a spirally fluted or braided appearance. Although differentiation in color patterns has been documented among species/populations, these nemerteans share similar external characters. Using the sequence datasets of mitochondrial cytochrome c oxidase subunit I (COI), 16S rRNA, and nuclear 28S rRNA genes of specimens from 14 localities of Canada, USA, Russia, Japan, and China, we analyzed the genetic differentiation and reconstructed the phylogenetic trees for these nemerteans. In conjunction with the external characters, we discuss their taxonomy and species delimitation. An analysis based on COI dataset showed high genetic variations among populations and even among worms from the same geographic area. The analyzed 111 individuals were assigned into seven networks by statistical parsimony analysis. The inter-network uncorrected p-distances ranged from 0.044 to 0.172 and the mean intra-network uncorrected p-distances varied from 0.001 to 0.005. With the exception of two networks that contain specimens from the East China Sea, all networks were well-supported by the results of Bayesian and neighbor-joining analyses on the COI data. Phylogenetic trees based on 16S rRNA and 28S rRNA datasets were basically similar to the COI trees, but specimens in some networks were merged into larger clades. Present molecular analyses support the validity of P. sanjuanensis and the synonymization of P. cylindracea with P. peregrina. Nemerteans previously recorded as P. peregrina may contain several species and sympatric speciation might have been occurred in this nemertean group.
Asunto(s)
ADN/genética , Invertebrados/clasificación , Invertebrados/genética , Animales , Clasificación , Variación Genética , FilogeniaRESUMEN
The complete mitochondrial genome of Iwatanemertes piperata (Nemertea: Heteronemertea) was determined. The genome, which contains 13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes, is 16,382 bp in length and has a base composition of G (25.87%), A (21.53%), T (40.64%) and C (11.95%). The gene order is identical to other Heteronemertea mitogenomes published to date.
Asunto(s)
Cestodos/genética , Genoma de los Helmintos , Genoma Mitocondrial , Animales , Secuencia de Bases , Cestodos/clasificación , Genes de ARNr , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , ARN de Transferencia/genética , Análisis de Secuencia de ADNRESUMEN
The cysts of nine Chinese populations of parthenogenetic Artemia were studied by scanning electron microscope. In the 270 cysts examined, 15 different morphological patterns were recognized with most of them not recorded in previous studies and the "tubercled shell surface" being the most common pattern. Results also displayed high intrapopulation variability, with the maximum of 11 patterns (in 30 cysts) recorded from the Barkol population. No positive correlation between the diversity of cyst shell patterns and ploidy compositions was found. Principal components analysis suggests higher similarity among coastal populations than among inland populations, which may be attributed to the identity of physicochemical conditions among coastal salterns and dissimilarity among inland saline lakes.
Asunto(s)
Artemia/ultraestructura , Óvulo/ultraestructura , Animales , Artemia/genética , Artemia/fisiología , China , Microscopía Electrónica de Rastreo , Ploidias , Análisis de Componente Principal , ReproducciónRESUMEN
We sequenced the complete mitochondrial genomes (mitogenomes) of three Hoplonemertea species, Amphiporus formidabilis, Prosadenoporus spectaculum and Nipponnemertes punctatula, which are 14,616, 14,655 and 15,354 bp in length, respectively. Each of the three circular mitogenomes consists of 37 typical genes and some non-coding regions. The nucleotide composition of the coding strand is biased toward T, almost a half of total nucleotides in these mitogenomes. There are many poly-T tracts across these mitogenomes, which exhibit T-number variation within different clones of protein-coding genes, mainly resulting from false PCR amplification. The major non-coding regions have tandem repeat motifs and hairpin-like structures that may be associated with the initiation of replication or transcription. Data published to date for nemerteans show that Palaeonemertea species usually bear the largest mitogenomes, while representatives in the more recently derived Distromatonemertea clade bear the smallest ones; and that the gene arrangement of mitogenomes seems to be variable within the phylum Nemertea, but stable within either of Heteronemertea and Hoplonemertea.
Asunto(s)
ADN de Helmintos/genética , ADN Mitocondrial/genética , Genoma Mitocondrial , Helmintos/clasificación , Helmintos/genética , Animales , Secuencia de Bases , Orden Génico , Genes de Helminto , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , ARN de Transferencia/genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Most nemerteans (phylum Nemertea) are free-living, but about 50 species are known to be firmly associated with other marine invertebrates. For example, Gononemertes parasita is associated with ascidians, and Nemertopsis tetraclitophila with barnacles. There are 12 complete or near-complete mitochondrial genome (mitogenome) sequences of nemerteans available in GenBank, but no mitogenomes of none free-living nemerteans have been determined so far. In the present paper complete mitogenomes of the above two parasitic/commensal nemerteans are reported. METHODS: The complete mitochondrial genomes (mitogenome) of G. parasita and N. tetraclitophila were amplified by conventional and long PCR. Phylogenetic analyses of maximum likelihood (ML) and Bayesian inference (BI) were performed with both concatenated nucleotide and amino acid sequences. RESULTS: Complete mitogenomes of G. parasita and N. tetraclitophila are 14742 bp and 14597 bp in size, respectively, which are within the range of published Hoplonemertea mitogenomes. Their gene orders are identical to that of published Hoplonemertea mitogenomes, but different from those of Palaeo- and Heteronemertea species. All the coding genes, as well as major non-coding regions (mNCRs), are AT rich, which is especially pronounced at the third codon position. The AT/GC skew pattern of the coding strand is the same among nemertean mitogenomes, but is variable in the mNCRs. Some slight differences are found between mitogenomes of the present species and other hoplonemerteans: in G. parasita the mNCR is biased toward T and C (contrary to other hoplonemerteans) and the rrnS gene has a unique 58-bp insertion at the 5' end; in N. tetraclitophila the nad3 gene starts with the ATT codon (ATG in other hoplonemerteans). Phylogenetic analyses of the nucleotide and amino acid datasets show early divergent positions of G. parasita and N. tetraclitophila within the analyzed Distromatonemertea species, and provide strong support for the close relationship between Hoplonemertea and Heteronemertea. CONCLUSION: Gene order is highly conserved within the order Monostilifera, particularly within the Distromatonemertea, and the special lifestyle of G. parasita and N. tetraclitophila does not bring significant variations to the overall structures of their mitogenomes in comparison with free-living hoplonemerteans.
Asunto(s)
Genoma Mitocondrial/genética , Invertebrados/genética , Animales , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Especificidad de la EspecieRESUMEN
We compared the anatomy of the holotype of the palaeonemertean Cephalothrix simula ( Iwata, 1952 ) with that of the holotypes of Cephalothrix hongkongiensis Sundberg, Gibson and Olsson, 2003 and Cephalothrix fasciculus ( Iwata, 1952 ), as well as additional specimens from Fukue (type locality of C. simula) and Hiroshima, Japan. While there was no major morphological discordance between these specimens, we found discrepancies between the actual morphology and some statements in the original description of C. simula with respect to supposedly species-specific characters. Our observation indicates that these three species cannot be discriminated by the anatomical characters so far used to distinguish congeners. For objectivity of scientific names, topogenetypes of the mitochondrial cytochrome c oxidase subunit I (COI) sequences are designated for C. simula, C. hongkongiensis, and C. fasciculus. Analysis of COI sequence showed that the Hiroshima population can be identified as C. simula, which has been found in previous studies from Trieste, Italy, and also from both the Mediterranean and Atlantic coasts of the Iberian Peninsula, indicating an artificial introduction via (1) ballast water, (2) ship-fouling communities, or (3) the commercially cultured oyster Crassostrea gigas ( Thunberg, 1793 ) brought from Japan to France in 1970s. Cephalothrix simula is known to be toxic, as it contains large amounts of tetrodotoxin (TTX). We report here that the grass puffer Takifugu niphobles ( Jordan and Snyder, 1901 )-also known to contain TTX- consumes C. simula. We suggest that the puffer may be able to accumulate TTX by eating C. simula.