Thermal optimality and physiological parameters inferred from experimental studies scale latitudinally with marine species occurrences.

Ocean warming is expected to occur due to anthropogenic climate change bringing a spatial shift of marine communities. Experimental data that characterize the aerobic power budget via an aerobic scope, thermal metabolic scope, or thermal preferences have been proposed as tools that can describe species distribution since they characterize species fitness or performance under different temperatures. This study tested the potential relationship between observed occurrences and different physiological studies in the Americas for 11 commercially important species in Mexico. Projections were also developed for Mexico's exclusive economic zone under different climate warming scenarios. The physiological data were fitted from optimum up to pejus temperatures and projected to sea surface temperatures for present (2003-2014) and Representative Concentration Pathway (RCP) scenarios (RCP 2.6, RCP 4.5, RCP 6.0, and RCP 8.5) for the period 2040-2050 and 2090-2100. For species with wide distributions in the Americas, the number of occurrences reported decreases at higher latitudes related to the decrease in species performance calculated from laboratory experiments. In addition, higher species occurrences are usually reported around optimum temperatures. Overall, the results suggest that pejus temperatures likely restrict latitudinal distribution, at least for widely distributed taxons. Regarding Mexican projections, the results varied widely by species. For example, in the Atlantic Ocean, Octopus maya and Panulirus argus are vulnerable to warming scenarios, while Centropomus undecimalis is not. Interestingly, northern Campeche Bank, the Gulf of California, and Western Baja California may act as thermal refugia for marine species indicating they could be assigned as protected areas to support fisheries throughout the Mexican exclusive economic zone. This research adds to the increasing evidence of the relationship between thermal niche and wild population distribution.

RpuS/R Is a Novel Two-Component Signal Transduction System That Regulates the Expression of the Pyruvate Symporter MctP in Sinorhizobium fredii NGR234.

The SLC5/STAC histidine kinases comprise a recently identified family of sensor proteins in two-component signal transduction systems (TCSTS), in which the signaling domain is fused to an SLC5 solute symporter domain through a STAC domain. Only two members of this family have been characterized experimentally, the CrbS/R system that regulates acetate utilization in Vibrio and Pseudomonas, and the CbrA/B system that regulates the utilization of histidine in Pseudomonas and glucose in Azotobacter. In an attempt to expand the characterized members of this family beyond the Gammaproteobacteria, we identified two putative TCSTS in the Alphaproteobacterium Sinorhizobium fredii NGR234 whose sensor histidine kinases belong to the SLC5/STAC family. Using reverse genetics, we were able to identify the first TCSTS as a CrbS/R homolog that is also needed for growth on acetate, while the second TCSTS, RpuS/R, is a novel system required for optimal growth on pyruvate. Using RNAseq and transcriptional fusions, we determined that in S. fredii the RpuS/R system upregulates the expression of an operon coding for the pyruvate symporter MctP when pyruvate is the sole carbon source. In addition, we identified a conserved DNA sequence motif in the putative promoter region of the mctP operon that is essential for the RpuR-mediated transcriptional activation of genes under pyruvate-utilizing conditions. Finally, we show that S. fredii mutants lacking these TCSTS are affected in nodulation, producing fewer nodules than the parent strain and at a slower rate.

Oviducal gland transcriptomics of Octopus maya through physiological stages and the negative effects of temperature on fertilization.

Background: Elevated temperatures reduce fertilization and egg-laying rates in the octopus species. However, the molecular mechanisms that control the onset of fertilization and egg-laying in the octopus' oviducal gland are still unclear; and the effect of temperature on the expression of key reproductive genes is unknown. This study aims to better understand the molecular bases of octopus fertilization and egg-laying, and how they are affected by elevated temperatures. Method: RNA-seq of oviducal glands was performed for samples before, during, and after fertilization and their transcriptomic profiles were compared. Also, at the fertilization stage, the optimal and thermal-stress conditions were contrasted. Expression levels of key reproductive genes were validated via RT-qPCR. Results: In mated females before egg-laying, genes required for the synthesis of spermine, spermidine, which may prevent premature fertilization, and the myomodulin neuropeptide were upregulated. Among the genes with higher expression at the fertilization stage, we found those encoding the receptors of serotonin, dopamine, and progesterone; genes involved in the assembly and motility of the sperm flagellum; genes that participate in the interaction between male and female gametes; and genes associated with the synthesis of eggshell mucoproteins. At temperatures above the optimal range for reproduction, mated females reduced the fertilization rate. This response coincided with the upregulation of myomodulin and APGW-amide neuropeptides. Also, genes associated with fertilization like LGALS3, VWC2, and Pcsk1 were downregulated at elevated temperatures. Similarly, in senescent females, genes involved in fertilization were downregulated but those involved in the metabolism of steroid hormones like SRD5A1 were highly expressed.

Novel viral RNA genomes of the vine mealybug Planococcus ficus.

The vine mealybug, Planococcus ficus (Signoret, 1875), is the most important insect pest in growing areas of the grapevine Vitis vinifera L. in several countries, including Mexico. In Mexico, Baja California (B.C.) is the region with the highest production of V. vinifera L. grapes for industrial purposes. Recently, the diversity of viruses infecting insects only (insect-specific viruses) has been broadly explored to elucidate further ecological viral-host interactions in many insect species, which in some cases has resulted in the application of virus-based biological control agents for insect pests. However, a survey of the Pl. ficus virome has not been done yet. In the present study, we pooled Pl. ficus individuals collected through different vineyards of Ensenada, B.C., Mexico and analysed them by meta-transcriptomics. Novel nearly complete genomes of five RNA viruses were retrieved. These viruses were related to the Iflaviridae and Reoviridae families, and to the Picornavirales and Tolivirales orders. A new isolate belonging to the Dicistroviridae family was also found. Phylogenetic analyses showed that these putative viral genomes group with viruses having hemipteran (including a mealybug species) or other insect hosts, or with viruses associated with insects. Our results suggest that the identified novel RNA viruses could be insect-specific viruses of Pl. ficus. This work is the first insight into the Pl. ficus virome; it guarantees further studies aimed to characterize those viruses with potential for application in biological control of this economically important insect.

Sex-specific role of the optic gland in octopus maya: A transcriptomic analysis.

The optic glands (OG) of cephalopods are a source of molecules associated with the control of reproductive traits and lifecycle events such as sexual maturation, reproductive behavior, feeding, parental care, and senescence. However, little is known about the role of the optic gland in Octopus maya adults during mating and egg laying. RNA sequencing, de novo transcriptome assembly, ubiquity and differential expression analysis were performed. First, we analyzed the expression patterns of transcripts commonly associated with OG regulatory functions to describe their possible role once the maturation of the gonad is complete. The transcriptomic profiles of the optic gland of both sexes were compared with emphasis on the signaling pathways involved in the dimorphism of reproductive traits. Results suggest that in the OG of males, the reproductive condition (mated or non-mated) did not affect the general expression profile. In contrast, more differentially expressed genes were observed in females. In mated females, the mRNA metabolic process and the response to norepinephrine were enriched, suggesting a high cellular activity in preparation for the laying of the embryos. Whereas in egg-laying females, energetic and metabolic processes were the most represented, including the oxidation-reduction process. Finally, the gene expression patterns in senescence females suggest a physiological response to starvation as well as upregulation of genes involved retrotransposon activity. In conclusion, more substantial fluctuations in gene expression were observed in the optic glands of the fertilized females compared to the males. Such differences might be associated with the regulation of the egg-laying and the onset of senescence.

Environmental conditions drive zooplankton community structure in the epipelagic oceanic water of the southern Gulf of Mexico: A molecular approach.

Zooplankton plays a pivotal role in sustaining the majority of marine ecosystems. The distribution patterns and diversity of zooplankton provide key information for understanding the functioning of these ecosystems. Nevertheless, due to the numerous cryptic and sibling species and the lack of diagnostic characteristics for early developmental stages, the identification of the global-to-local patterns of zooplankton biodiversity and biogeography remains challenging in different research fields. The spatial and temporal changes in the zooplankton community in the open waters of the southern Gulf of Mexico were assessed using metabarcoding analysis of the V9 region of 18S rRNA and mitochondrial cytochrome oxidase c subunit I (COI). Additionally, a multiscale analysis was implemented to evaluate which environmental predictors may explain the variability in the structure of the zooplankton community. Our findings suggest that the synergistic effects of dissolved oxygen concentration, temperature, and longitude (intended as a proxy for still unidentified predictors) may explain both spatial and temporal zooplankton variability even with low contribution. Furthermore, the zooplankton distribution probably reflects the coexistence of three heterogeneous ecoregions and a bio-physical partitioning of the studied area. Finally, some taxa were either exclusive or predominant with either 18S or COI markers. This may suggest that comprehensive assessments of the zooplankton community may be more accurately met by the use of multilocus approaches.

Transcriptomic assessment of dietary fishmeal partial replacement by soybean meal and prebiotics inclusion in the liver of juvenile Pacific yellowtail (Seriola lalandi).

BACKGROUND: Seriola lalandi is an important species for aquaculture, due to its rapid growth, adaptation to captivity and formulated diets, and high commercial value. Due to the rise in fishmeal (FM) price, efforts have been and still are made to replace it partially or entirely with vegetable meals in diets for carnivorous fish. The use of prebiotics when feeding vegetable meals has improved fish health. METHODS: Four experimental diets were assessed in juveniles, the control diet consisted of FM as the main protein source, the second diet included 2% of GroBiotic®-A (FM-P), in the third diet FM was partially replaced (25%) by soybean meal (SM25), and the fourth consisted of SM25 with 2% of GroBiotic®-A (SM25-P). Growth was evaluated and RNA-seq of the liver tissue was performed, including differential expression analysis and functional annotation to identify genes affected by the diets. RESULTS: Growth was not affected by this level of FM replacement, but it was improved by prebiotics. Annotation was achieved for 59,027 transcripts. Gene expression was affected by the factors: 225 transcripts due to FM replacement, 242 due to prebiotics inclusion, and 62 due to the interaction of factors. The SM25-P diet showed the least amount of differentially expressed genes against the control diet. CONCLUSION: The replacement of FM (25%) by soybean meal combined with prebiotics (2%) represents a good cost-benefit balance for S. lalandi juveniles since the fish growth increased and important metabolic and immune system genes in the liver were upregulated with this diet.

Relationship between paralytic shellfish toxin content and sxtA gene copy number in different growth phases of Gymnodinium catenatum (Dinophyceae).

Paralytic shellfish toxin (PST) content in the dinoflagellate Gymnodinium catenatum changes with culture age, with a higher toxin concentration in the logarithmic phase that decreases when the culture ages. The gene copy number (GCN) of domains sxtA1 and sxtA4 was higher in the lag and stationary phase, and lag phase, respectively. No relationship was found between the GCN of the domains sxtA4 and sxtA1 with the PST content in G. catenatum.

Acidification stress effect on umbonate veliger larval development in Panopea globosa.

Ocean acidification generates a decrease in calcium carbonate availability essential for biomineralization in organisms such as mollusks. This effect was evaluated on Panopea globosa exposing for 7 days umbonate veliger larvae to two pH treatments: experimental (pH 7.5) and control (pH 8.0). Exposure to pH 7.5 affected growth, reducing larval shell length from 5.15-13.34% compared to the control group. This size reduction was confirmed with electron microscopy, also showing shell damage. The physiological response showed an increase in oxygen consumption in larvae exposed to low pH with a maximum difference of 1.57 nmol O2 h-1 larvae-1 at day 7. The gene expression analyses reported high expression values for nicotinamide adenine dinucleotide (NADH) dehydrogenase and Perlucin in larvae at pH 7.5, suggesting a higher energetic cost in this larval group to maintain homeostasis. In conclusion, this study showed that acidification affected development of P. globosa umbonate veliger larvae.

Transcriptomic response and hydrocarbon accumulation in the eastern oyster (Crassostrea virginica) exposed to crude oil.

The adverse effect of crude oil on marine invertebrates is well known. To have a better understanding of its effects on marine invertebrates, Crassostrea virginica was exposed to different concentrations (50, 100 and 200 µg/L) of a mixture of super-light and light crude oil for two weeks, evaluating the transcriptomic response of the digestive gland using RNA-Seq and their accumulation in soft tissues. A total of 33,469,374 reads were assembled, which resulted in 61,356 genome assemblies ('Genes'). Trinotate was used for transcript annotation. At the end of this process, 86,409 transcripts were maintained, comprising a broad set of enzymes from xenobiotics metabolism, oxidative stress, stress and immune responses, and energetic metabolism. The enrichment analysis revealed a change in biological processes and molecular functions, finding from 100 to 200 µg/L. Moreover, the differential gene expression analysis showed a dose-dependent transcriptional response, generally up to 100 µg/L and in some cases up to 200 µg/L, which suggested that oysters' response decreased after 100 µg/L; the analysis of crude oil presence in soft tissues indicated that C. virginica is a suitable candidate for ecotoxicology. Finally, these results should contribute to expanding current genomic resources for C. virginica. Furthermore, they will help to develop new studies in aquatic toxicology focused on knowledge in depth of metabolic pathways, jointly with other approaches (such as proteomics) to allow obtaining a complete idea about the eastern oyster response to crude oil.

Correction to: Aspergillus loretoensis, a single isolate from marine sediment of Loreto Bay, Baja California Sur, México resulting as a new obligate halophile species.

In the original publication the Figs. 3 and 4 are published inappropriately. The correct version of Figs. 3 and 4 is provided in this correction.

Aspergillus loretoensis, a single isolate from marine sediment of Loreto Bay, Baja California Sur, México resulting as a new obligate halophile species.

An obligate halophile fungal was isolated from 275 m deep marine sediments and is characterized here for the first time. Its optimal growth was at 15% NaCl even though it was able to grow at 25% and is incapable of growth with no NaCl. Based on its morphological characteristics as conidia chain production in a single phialide, the fungal is related to the genus Aspergillus, subgenus Polypaecilum. Phylogenetic molecular analysis using several markers (ITS1-2; RPB1; RPB2; Cct8; TSR1; CaM; BenA) places the fungal isolate closer to Aspergillus salinarus and A. baarnensis. However, its morphological and molecular differences establish it as a new species, Aspergillus loretoensis sp. nov.

Octopus maya white body show sex-specific transcriptomic profiles during the reproductive phase, with high differentiation in signaling pathways.

White bodies (WB), multilobulated soft tissue that wraps the optic tracts and optic lobes, have been considered the hematopoietic organ of the cephalopods. Its glandular appearance and its lobular morphology suggest that different parts of the WB may perform different functions, but a detailed functional analysis of the octopus WB is lacking. The aim of this study is to describe the transcriptomic profile of WB to better understand its functions, with emphasis on the difference between sexes during reproductive events. Then, validation via qPCR was performed using different tissues to find out tissue-specific transcripts. High differentiation in signaling pathways was observed in the comparison of female and male transcriptomic profiles. For instance, the expression of genes involved in the androgen receptor-signaling pathway were detected only in males, whereas estrogen receptor showed higher expression in females. Highly expressed genes in males enriched oxidation-reduction and apoptotic processes, which are related to the immune response. On the other hand, expression of genes involved in replicative senescence and the response to cortisol were only detected in females. Moreover, the transcripts with higher expression in females enriched a wide variety of signaling pathways mediated by molecules like neuropeptides, integrins, MAPKs and receptors like TNF and Toll-like. In addition, these putative neuropeptide transcripts, showed higher expression in females' WB and were not detected in other analyzed tissues. These results suggest that the differentiation in signaling pathways in white bodies of O. maya influences the physiological dimorphism between females and males during the reproductive phase.

DNA barcoding and morphological taxonomy: identification of lanternfish (Myctophidae) larvae in the Gulf of Mexico.

Accurate identification of fish larval stages is complicated and time-consuming due to the lack of diagnostic morphological characters, especially during early developmental stages. The distribution of lanternfish (Myctophidae) has been described based on the morphological identification of adult stages. Larvae of only a few species of Myctophidae have been described, and the description is not always precise. In this study, larvae were collected and morphologically identified as Diaphus mollis, Hygophum hygomii, H. reinhardtii, H. taaningi, Myctophum obtusirostre and M. selenops. The DNA barcode region of the mitochondrial cytochrome oxidase I gene (COI) was determined for all larvae. The COI sequences matched reference barcodes available in GenBank for 14 of the identified larvae. The remaining COI sequences matched reference barcodes for different species of Myctophidae including Centrobranchus nigroocellatus, Diogenichthys atlanticus and Lepidophanes guentheri. This effort demonstrated the importance of integrated morphological and molecular analysis of species diversity and distribution of the Myctophidae in the Gulf of México.

Transcriptomic and metabolic response to chronic and acute thermal exposure of juvenile geoduck clams Panopea globosa.

The Cortes geoduck Panopea globosa is a large bivalve with a high commercial value distributed from the southern Pacific coast of the Baja California Peninsula to the northern Gulf of California, inhabiting a wide range of subtropical temperatures. A new record of this species in shallower waters suggests that it can tolerate a warmer environment than previously thought. To better understand the whole-body and molecular response mechanisms to different temperatures, we assessed the metabolic rate of juvenile individuals exposed to chronic and acute thermal conditions and analyzed the transcriptomic response in ctenidial tissues. Whole-body metabolic rate measurements showed that juveniles were able to acclimate at least partially within three weeks from 20 °C (C20) to 29 °C (C29), while organisms acutely exposed to 29 °C (A29) significantly increased their metabolic rate. This was coincident with transcriptomic results, as similar gene expression patterns were found in clams chronically exposed to C29 and C20, but different from those acutely exposed to 29 °C (A29) and 31 °C (A31). High degree of expression of genes involved in DNA repair and transcription regulation were found in C29 juveniles, whereas protective genes against oxidative stress were highly expressed in A29 organisms. A high expression of genes involved in protein re-folding was also observed in A31 juveniles. In conclusion, the combined results of whole-body metabolism and transcriptomic expression patterns suggest that P. globosa juveniles have a high physiological plasticity and are well adapted to inhabit an environment with broad temperature fluctuations.

Transcriptomic Analysis Reveals Insights on Male Infertility in Octopus maya Under Chronic Thermal Stress.

Octopus maya endemic to the Yucatan Peninsula, Mexico, is an ectotherm organism particularly temperature-sensitive. Studies in O. maya females show that temperatures above 27°C reduce the number of eggs per spawn, fertilization rate and the viability of embryos. High temperatures also reduce the male reproductive performance and success. However, the molecular mechanisms are still unknown. The transcriptomic profiles of testes from thermally stressed (30°C) and not stressed (24°C) adult male octopuses were compared, before and after mating to understand the molecular bases involved in the low reproductive performance at high temperature. The testis paired-end cDNA libraries were sequenced using the Illumina MiSeq platform. Then, the transcriptome was assembled de novo using Trinity software. A total of 53,214,611 high-quality paired reads were used to reconstruct 85,249 transcripts and 77,661 unigenes with an N50 of 889 bp length. Later, 13,154 transcripts were annotated implementing Blastx searches in the UniProt database. Differential expression analysis revealed 1,881 transcripts with significant difference among treatments. Functional annotation and pathway mapping of differential expressed transcripts revealed significant enrichment for biological processes involved in spermatogenesis, gamete generation, germ cell development, spermatid development and differentiation, response to stress, inflammatory response and apoptosis. Remarkably, the transcripts encoding genes such as ZMYND15, KLHL10, TDRD1, TSSK2 and DNAJB13, which are linked to male infertility in other species, were differentially expressed among the treatments. The expression levels of these key genes, involved in sperm motility and spermatogenesis were validated by quantitative real-time PCR. The results suggest that the reduction in male fertility at high temperature can be related to alterations in spermatozoa development and motility.

Transcriptomic information from Pacific white shrimp (Litopenaeus vannamei) ovary and eyestalk, and expression patterns for genes putatively involved in the reproductive process.

The increased use of massive sequencing technologies has enabled the identification of several genes known to be involved in different mechanisms associated with reproduction that so far have only been studied in vertebrates and other model invertebrate species. In order to further investigate the genes involved in Litopenaeus vannamei reproduction, cDNA and SSH libraries derived from female eyestalk and gonad were produced, allowing the identification of expressed sequences tags (ESTs) that potentially have a role in the regulation of gonadal maturation. In the present study, different transcripts involved in reproduction were identified and a number of them were characterized as full-length. These transcripts were evaluated in males and females in order to establish their tissue expression profiles during developmental stages (juvenile, subadult and adult), and in the case of females, their possible association with gonad maturation was assessed through expression analysis of vitellogenin. The results indicated that the expression of vitellogenin receptor (vtgr) and minichromosome maintenance (mcm) family members in the female gonad suggest an important role during previtellogenesis. Additionally, the expression profiles of genes such as famet, igfbp and gpcr in brain tissues suggest an interaction between the insulin/insulin-like growth factor signaling pathway (IIS) and methyl farnesoate (MF) biosynthesis for control of reproduction. Furthermore, the specific expression pattern of farnesoic acid O-methyltransferase suggests that final synthesis of MF is carried out in different target tissues, where it is regulated by esterase enzymes under a tissue-specific hormonal control. Finally, the presence of a vertebrate type steroid receptor in hepatopancreas and intestine besides being highly expressed in female gonads, suggest a role of that receptor during sexual maturation.

Global transcriptional analysis suggests Lasiodiplodia theobromae pathogenicity factors involved in modulation of grapevine defensive response.

BACKGROUND: Lasiodiplodia theobromae is a fungus of the Botryosphaeriaceae that causes grapevine vascular disease, especially in regions with hot climates. Fungi in this group often remain latent within their host and become virulent under abiotic stress. Transcriptional regulation analysis of L. theobromae exposed to heat stress (HS) was first carried out in vitro in the presence of grapevine wood (GW) to identify potential pathogenicity genes that were later evaluated for in planta expression. RESULTS: A total of 19,860 de novo assembled transcripts were obtained, forty-nine per cent of which showed homology to the Botryosphaeriaceae fungi, Neofusicoccum parvum or Macrophomina phaseolina. Three hundred ninety-nine have homology with genes involved in pathogenic processes and several belonged to expanded gene families in others fungal grapevine vascular pathogens. Gene expression analysis showed changes in fungal metabolism of phenolic compounds; where genes encoding for enzymes, with the ability to degrade salicylic acid (SA) and plant phenylpropanoid precursors, were up-regulated during in vitro HS response, in the presence of GW. These results suggest that the fungal L-tyrosine catabolism pathway could help the fungus to remove phenylpropanoid precursors thereby evading the host defense response. The in planta up-regulation of salicylate hydroxylase, intradiol ring cleavage dioxygenase and fumarylacetoacetase encoding genes, further supported this hypothesis. Those genes were even more up-regulated in HS-stressed plants, suggesting that fungus takes advantage of the increased phenylpropanoid precursors produced under stress. Pectate lyase was up-regulated while a putative amylase was down-regulated in planta, this could be associated with an intercellular growth strategy during the first stages of colonization. CONCLUSIONS: L. theobromae transcriptome was established and validated. Its usefulness was demonstrated through the identification of genes expressed during the infection process. Our results support the hypothesis that heat stress facilitates fungal colonization, because of the fungus ability to use the phenylpropanoid precursors and SA, both compounds known to control host defense.

Quantitative real-time PCR normalization for gene expression studies in the plant pathogenic fungi Lasiodiplodia theobromae.

Lasiodiplodia theobromae is a highly virulent plant pathogen. It has been suggested that heat stress increases its virulence. The aim of this work was to evaluate, compare, and recommend normalization strategies for gene expression analysis of the fungus growing with grapevine wood under heat stress. Using RT-qPCR-derived data, reference gene stability was evaluated through geNorm, NormFinder and Bestkeeper applications. Based on the geometric mean using the ranking position obtained for each independent analysis, genes were ranked from least to most stable as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), ß-tubulin (TUB) and elongation factor-1α (EF1α). Using RNAseq-derived data based on the calculated tagwise dispersion these genes were ordered by increasing stability as follows: GAPDH, ACT, TUB, and EF1α. The correlation between RNAseq and RTqPCR results was used as criteria to identify the best RT-qPCR normalization approach. The gene TUB is recommended as the best option for normalization among the commonly used reference genes, but alternative fungal reference genes are also suggested.

Behavioral thermoregulation and critical thermal limits of giant keyhole limpet Megathura crenulata(Sowerby 1825) (Mollusca; Vetigastropoda).

The thermoregulatory behavior of the giant keyhole limpet Megathura crenulata was determined in a horizontal thermal gradient during the day at 18.9 °C and 18.3 °C for the night. The final preferendum determined for giant keyhole limpets was of 18.6±1.2 °C. Limpets' displacement velocity was 10.0±3.9 cm h(-1) during the light phase and 8.4±1.6 cm h(-1) during the dark phase. The thermotolerance (measured as CTMax at 50%) was determined in a keyhole limpet in three acclimation temperatures 17, 20, and 23 °C. Limpets were subjected to water increasing temperatures at a rate of 1 °C every 30 min, until they detached from the substrate. The critical thermal maximum at 50% was 27.2, 27.9 and 28.3 °C respectively.