Chitin isolation from crustacean waste using a hybrid demineralization/DBD plasma process.

Conventional isolation of chitin from crustacean waste demands the use of high amounts of hazardous chemicals, hence not leading to a sustainable process. Atmospheric-pressure dielectric barrier discharge (DBD) plasma has demonstrated an enhanced ability to remove proteins directly from the biomass without the formation of any waste. Simultaneously, organic acids have proven very efficient in the removal of inorganic minerals from crustacean waste. Therefore, a hybrid process composed of DBD plasma and demineralization using organic acids has been successfully applied for the isolation of chitin. Results showed that the integration of nitrogen-based plasma and lactic acid demineralization allowed the elimination of 90 % of the proteins and ensures the complete removal of minerals from shrimp shells waste. The isolated chitin was further characterized using distinct techniques, namely XPS, ATR-FTIR, XRD and SEM. Chitin degree of deacetylation and molecular weight were also assessed. Hence, this work presents a sustainable and feasible platform for the extraction and purification of chitin from crustacean waste with almost zero waste formed.

Toxicology study of fraxinellone as ovicidal agents against Mythimna separata Walker and Bombyx mori Linaeus.

Fraxinellone is a naturally occurring degraded limonoid isolated from many species of plants in Meliaceae and Rutaceae. Besides structural modification of the lead compounds, the toxicology study of the lead compounds is also a very important procedure to develop insecticidal agents. Herein the toxicology study of fraxinellone was carried out as the ovicidal agent against the eggs of two lepidopteran insects Mythimna separata Walker and Bombyx mori Linaeus. Fraxinellone selectively exhibited an ovicidal activity against the eggs of M. separata. After treatment with fraxinellone, the eggshells of M. separata were shrinked, whereas those of B. mori had no obvious change. The dynamic process of M. separata embryo development demonstrated that the distinct difference between the treated eggs and the control ones was obvious at the second day after treatment, especially, the control embryo finished blastokinesis, whereas the treated ones were still laid at pre-reversion status and a lot of yolk can be seen around the embryo. It ultimately resulted in the eggshell withered and the egg hatching inhibited.

Water wettability dependence on surface structure of a snail shell.

There are many reports on the special wettability of hierarchical surface structures in nature. Snail shells with three types of roughness of 10, 100, and 500 µm have a unique wetting behavior. In the present study, we investigate the influence of the surface structure on the water wettability using snail shells with different surface roughness. The wettability of a water droplet on the samples was evaluated. The three types of roughness on the surface structure of snail shell had higher water droplet spreading properties than the two types of roughness 500 µm and, 10 or 100 µm. Surface structures of snail shells with different surface roughness were simulated using epoxy resins to clarify the mechanism for the dynamics wetting behavior. The contact angle with a hydrophobic nature, of the epoxy resin with the three types of roughness decreased with increasing time, indicating a hydrophilic nature. The base diameter of the epoxy resins with the three types of roughness increased with increasing time. This was larger than that for a flat epoxy resin with hydrophilicity. Other epoxy resins with shell texture containing 100 and 500 or 10 and 500 µm roughness showed almost no change in the contact angle and diameter of the droplet base. The three types of roughness on the sample surface contributed to development of the water droplet spreading. The 10 µm roughness of the sample surface influenced the dynamic contact angles.

Recoverable impacts of ocean acidification on the tubeworm, Hydroides elegans: implication for biofouling in future coastal oceans.

Ocean uptake of anthropogenic CO2 causes ocean acidification (OA), which not only decreases the calcification rate, but also impairs the formation of calcareous shells or tubes in marine invertebrates such as the dominant biofouling tubeworm species, Hydroides elegans. This study examined the ability of tubeworms to resume normal tube calcification when returned to ambient pH 8.1 from a projected near-future OA level of pH 7.8. Tubeworms produced structurally impaired and mechanically weaker calcareous tubes at pH 7.8 compared to at pH 8.1, but were able to recover when the pH was restored to ambient levels. This suggests that tubeworms can physiologically recover from the impacts of OA on tube calcification, composition, density, hardness and stiffness when returned to optimal conditions. These results help understanding of the progression of biofouling communities dominated by tubeworms in future oceans with low pH induced by OA.

Biomonitoring acidification using marine gastropods.

Ocean acidification is mainly being monitored using data loggers which currently offer limited coverage of marine ecosystems. Here, we trial the use of gastropod shells to monitor acidification on rocky shores. Animals living in areas with highly variable pH (8.6-5.9) were compared with those from sites with more stable pH (8.6-7.9). Differences in site pH were reflected in size, shape and erosion patterns in Nerita chamaeleon and Planaxis sulcatus. Shells from acidified sites were shorter, more globular and more eroded, with both of these species proving to be good biomonitors. After an assessment of baseline weathering, shell erosion can be used to indicate the level of exposure of organisms to corrosive water, providing a tool for biomonitoring acidification in heterogeneous intertidal systems. A shell erosion ranking system was found to clearly discriminate between acidified and reference sites. Being spatially-extensive, this approach can identify coastal areas of greater or lesser acidification. Cost-effective and simple shell erosion ranking is amenable to citizen science projects and could serve as an early-warning-signal for natural or anthropogenic acidification of coastal waters.

Behavioural Responses of Unio tumidus Freshwater Mussels to Pesticide Contamination.

A pesticide is a chemical substance used for the disposal of pests, such as insects, weeds, invertebrates, or rodents. Pesticides interfere with the normal metabolism of the target species; however, some of them may inadvertently affect organisms other than those targeted. Increased quantities of pesticides in water disturb various ecological processes and may increase the mortality rate of various native species of flora and fauna. One of the groups of organisms that are at the greatest risk from the adverse effects of pesticides is the bivalves. This study was designed to assess the behavioural reaction of bivalves to widespread pesticides. As a representative example, the Polish native Unio tumidus (Philipsson 1788) was used. The study investigated different groups of toxic pesticides, such as herbicides (lenacil), insecticides (thiacloprid, DDT and dichlorvos), and fungicides (tebuconazole), in concentrations of 10 mg L-1. The results showed various behavioural reactions of bivalves to the pesticides. The most evident were activity time and shell opening rate. Moreover, as a result of DDVP contamination, effects were recorded in terms of shell opening level as well as rapid onset of death. Among the five analysed plant protection products, the most toxic was DDVP. Its presence caused adductor muscle paralysis in all analysed individuals. The least toxic pesticides were DDT and thiacloprid. A strong reaction to lenacil was observed especially in the shell opening rate. Tebuconazole caused significant reductions in activity. Despite the fact that the impact of pesticides on ecosystems is under regular observation, with the use of a wide range of scientific techniques, the use of bivalves was shown to have considerable potential for water quality monitoring.

Impairment of the immune response after transcuticular introduction of the insect gonadoinhibitory and hemocytotoxic peptide Neb-colloostatin: A nanotech approach for pest control.

This article shows that nanodiamonds can transmigrate through the insect cuticle easily, and the doses used were not hemocytotoxic and did not cause inhibition of cellular and humoral immune responses in larvae, pupae and adults of Tenebrio molitor. The examination of the nanodiamond biodistribution in insect cells demonstrated the presence of nanodiamond aggregates mainly in hemocytes, where nanoparticles were efficiently collected as a result of phagocytosis. To a lesser extent, nanodiamond aggregates were also detected in fat body cells, while they were not observed in Malpighian tubule cells. We functionalized nanodiamonds with Neb-colloostatin, an insect hemocytotoxic and gonadoinhibitory peptide, and we showed that this conjugate passed through the insect cuticle into the hemolymph, where the peptide complexed with the nanodiamonds induced apoptosis of hemocytes, significantly decreased the number of hemocytes circulating in the hemolymph and inhibited cellular and humoral immune responses in all developmental stages of insects. The results indicate that it is possible to introduce a peptide that interferes with the immunity and reproduction of insects to the interior of the insect body by means of a nanocarrier. In the future, the results of these studies may contribute to the development of new pest control agents.

Changes in exoskeleton surface roughness and expression of chitinase genes in mud crab Macrophthalmus japonicus following heavy metal differences of estuary.

Risk assessment of heavy metals is important for the health evaluation of inhabiting species in aquatic ecosystem. This study investigated whether chitin exoskeleton of mud crab Macrophthalmus japonicus is affected by heavy metals in estuary sediments in Korea. We compared heavy metal concentrations and analyzed the expression of M. japonicus chitinase genes, which play the crucial role in the formation of chitin exoskeleton. Concentrations of heavy metals were highly observed in crab body inhabiting Hampyeong among estuarine sites. High expressions of chitinase 1 were observed in crab gill and hepatopancreas from Myodo, which is the site with the lowest concentration of heavy metal in crab body. The surface roughness of the exoskeleton decreased with the increased concentration of heavy metals accumulated in the crab body. These results suggest that the total bioconcentration of heavy metals in crabs affected the expression of chitinase genes and changes in the exoskeleton surface roughness.

Energy metabolism and survival of the juvenile recruits of the American lobster (Homarus americanus) exposed to a gradient of elevated seawater pCO2.

The transition from the last pelagic larval stage to the first benthic juvenile stage in the complex life cycle of marine invertebrates, such as the American lobster Homarus americanus, a species of high economic importance, represents a delicate phase in these species development. Under future elevated pCO2 conditions, ocean acidification and other elevated pCO2 events can negatively affect crustaceans. This said their effects on the benthic settlement phase are virtually unknown. This study aimed to identify the effects of elevated seawater pCO2 on stage V American lobsters exposed to seven pCO2 levels. The survival, development time, metabolic and feeding rates, carapace composition, and energy metabolism enzyme function were investigated. Results suggested an increase in mortality, slower development and an increase in aerobic capacity with increasing pCO2. Our study points to potential reduction in juvenile recruitment success as seawater pCO2 increases, thus foreshadowing important socio-economic repercussions for the lobster fisheries and industry.

Trace element biomineralisation in the carapace in male and female Argulus japonicus.

Parasites of fishes have been shown to be effective bioindicators of the aquatic environment. Few investigations have been conducted on ectoparasite models and therefore little is known about the fate of trace elements and metals which they accumulate. In this study trace element sequestration was observed in the carapace of the fish louse, Argulus japonicus and found to relate to the sex of the parasite, as well as, the degree of sclerotization of the carapace. Adults of A. japonicus were collected from cyprinid hosts in the Vaal Dam, South Africa. Parasites were removed and flash frozen in liquid nitrogen before being sectioned with a cryomicrotome. Sections and whole mounts of parasites were prepared and treated with Phen-Green TM FL cell-permeant diacetate. Cryosections were assessed for trace elements and metals using a scanning electron microscope equipped with energy dispersive spectroscopy. Results indicated that in both male and female parasites, trace elements become bound to the carapace and produce more intense fluorescence than in soft tissues. Sexual dimorphic differences were further observed between male and female parasites. The intensity of the fluorescence signals was greater in the carapace of male parasites than in females, particularly when comparing the carapace of the ventral side of the thorax. In females, an amorphous layer of material surrounding the eggs was observed and produced an intense fluorescent signal. Levels of trace elements and metals detected were not significantly different between male and female parasites. Results observed serve as a demonstration for the first time of trace element sequestration in a freshwater crustacean parasite and possible mechanisms employed to reduce body burdens of trace elements and metals.

Seasonal variations of contamination and exoskeletal malformations in the white shrimps Palaemon longirostris in the Gironde estuary, France.

Since the end of the 1980s, white shrimps (Palaemon longirostris) from the Gironde estuary have exhibited exoskeletal malformations, mainly involving cephalothorax, rostrum, scaphocerites and uropods. An 8-month study was carried out in 2015. Each month, 200 individuals were sampled and examined for exoskeletal malformations. Temporal variations in malformation frequency were noted, particularly during the breeding period, along with decreases in the size of non-deformed shrimps related to the appearance of juveniles in breeding sites, and high mortality among deformed shrimps. A significant increase in proportions of deformed shrimp was observed, relating particularly to the size (and therefore the age) of individuals. No significant difference was found between shrimp proportions with different numbers of malformations (one to four) for a fixed size class, nor was there any variation in proportions within different size classes for a fixed number of malformations. This would appear to indicate that the number of malformations is acquired and new malformations do not seem to appear during the life cycle, except for the smallest (youngest) shrimps. The malformation spectrum showed no significant differences between the biggest and smallest individuals for the different malformation associations, except for those involving cephalothorax, rostrum and uropods. This would suggest that some malformation associations lead to a higher mortality rate in shrimps subjected to them, due to greater impairment of feeding and/or swimming behaviour. Multiple component analysis of the different types of malformation showed correlations between exoskeletal pieces (rostrum and cephalothorax) and appendixes (scaphocerites and uropods). Regarding metal contamination in shrimp, no significant difference was highlighted between deformed and non-deformed shrimps. Organic pollutants were not measured in tissues. Certain herbicides such as metolachlore and chlortoluron were detected at high concentrations in the Gironde estuary during the breeding period corresponding to the higher occurrence of exoskeletal malformations.

Bivalve shell formation in a naturally CO2-enriched habitat: Unraveling the resilience mechanisms from elemental signatures.

Marine bivalves inhabiting naturally pCO2-enriched habitats can likely tolerate high levels of acidification. Consequently, elucidating the mechanisms behind such resilience can help to predict the fate of this economically and ecologically important group under near-future scenarios of CO2-driven ocean acidification. Here, we assess the effects of four environmentally realistic pCO2 levels (900, 1500, 2900 and 6600 µatm) on the shell production rate of Mya arenaria juveniles originating from a periodically pCO2-enriched habitat (Kiel Fjord, Western Baltic Sea). We find a significant decline in the rate of shell growth as pCO2 increases, but also observe unchanged shell formation rates at moderate pCO2 levels of 1500 and 2900 µatm, the latter illustrating the capacity of the juveniles to partially mitigate the impact of high pCO2. Using recently developed geochemical tracers we show that M. arenaria exposed to a natural pCO2 gradient from 900 to 2900 µatm can likely concentrate HCO3- in the calcifying fluid through the exchange of HCO3-/Cl- and simultaneously maintain the pH homeostasis through active removal of protons, thereby being able to sustain the rate of shell formation to a certain extent. However, with increasing pCO2 beyond natural maximum the bivalves may have limited capacity to compensate for changes in the calcifying fluid chemistry, showing significant shell growth reduction. Findings of the present study may pave the way for elucidating the underlying mechanisms by which marine bivalves acclimate and adapt to high seawater pCO2.

Triangular gold nanoparticles modify shell characteristics and increase antioxidant enzyme activities in the clam Ruditapes decussatus.

CONTEXT: Nanoparticles may cause adverse environmental effects but there is limited information on their interactions with marine organisms. OBJECTIVE: Our aim was to examine the effects of triangular gold nanoparticles (Tr-Au NPs) on the clam, Ruditapes decussatus. MATERIALS AND METHODS: Clams were exposed to Tr-Au1 = 5 µg/L and Tr-Au2 = 10 µg/L for 2 and 7 days. Effects on shell structure were investigated. Superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST) activities, protein carbonyl levels and malondialdehyde content were used to assess biochemical status. RESULTS: Transmission electron microscopy (TEM) and electron dispersive X-ray microanalysis (EDX) showed that Tr-Au NPs modified shell structure and morphology. Tr-Au NPs size increased forming aggregate particles. Tr-Au NPs increased SOD, CAT and GST activities in gill and digestive gland in a concentration- and time-dependent manner indicating defence against oxidative stress. Enhanced lipid peroxidation and protein carbonyl levels confirmed oxidative stress. CONCLUSION: Tr-Au NPs cause oxidative stress and affect shell structure of clams. These findings may have relevance to other marine species.

The physiological effects of oil, dispersant and dispersed oil on the bay mussel, Mytilus trossulus, in Arctic/Subarctic conditions.

Increasing oil development around Alaska and other Arctic regions elevates the risk for another oil spill. Dispersants are used to mitigate the impact of an oil spill by accelerating natural degradation processes, but the reduced hydrophobicity of dispersed oil may increase its bioavailability to marine organisms. There is limited research on the effect of dispersed oil on cold water species and ecosystems. Therefore, spiked exposure tests were conducted with bay mussels (Mytilus trossulus) in seawater with non-dispersed oil, Corexit 9500 and oil dispersed with different concentrations of Corexit 9500. After three weeks of exposure, acute and chronic physiological impacts were determined. The majority of physiological responses occurred during the first seven days of exposure, with mussels exhibiting significant cytochrome P450 activity, superoxide dismutase activity and heat shock protein levels. Mussels exposed to non-dispersed oil also experienced immune suppression, reduced transcription and higher levels of mortality. After 21 days, mussels in all treatments exhibited evidence of genetic damage, tissue loss and a continued stress response. Bay mussels are useful as indicators of ecosystem health and recovery, and this study was an important step in understanding how non-dispersed oil, dispersant and dispersed oil affect the physiology of this sentinel species in Arctic/subarctic conditions.

Gene expression correlated with delay in shell formation in larval Pacific oysters (Crassostrea gigas) exposed to experimental ocean acidification provides insights into shell formation mechanisms.

BACKGROUND: Despite recent work to characterize gene expression changes associated with larval development in oysters, the mechanism by which the larval shell is first formed is still largely unknown. In Crassostrea gigas, this shell forms within the first 24 h post fertilization, and it has been demonstrated that changes in water chemistry can cause delays in shell formation, shell deformations and higher mortality rates. In this study, we use the delay in shell formation associated with exposure to CO2-acidified seawater to identify genes correlated with initial shell deposition. RESULTS: By fitting linear models to gene expression data in ambient and low aragonite saturation treatments, we are able to isolate 37 annotated genes correlated with initial larval shell formation, which can be categorized into 1) ion transporters, 2) shell matrix proteins and 3) protease inhibitors. Clustering of the gene expression data into co-expression networks further supports the result of the linear models, and also implies an important role of dynein motor proteins as transporters of cellular components during the initial shell formation process. CONCLUSIONS: Using an RNA-Seq approach with high temporal resolution allows us to identify a conceptual model for how oyster larval calcification is initiated. This work provides a foundation for further studies on how genetic variation in these identified genes could affect fitness of oyster populations subjected to future environmental changes, such as ocean acidification.

Chronic exposure of a freshwater mussel to elevated pCO2 : Effects on the control of biomineralization and ion-regulatory responses.

Freshwater mussels may be exposed to elevations in mean partial pressure of carbon dioxide (pCO2 ) caused by both natural and anthropogenic factors. The goal of the present study was to assess the effects of a 28-d elevation in pCO2 at 15 000 and 50 000 µatm on processes associated with biomineralization, ion regulation, and cellular stress in adult Lampsilis siliquoidea (Barnes, 1823). In addition, the capacity for mussels to compensate for acid-base disturbances experienced after exposure to elevated pCO2 was assessed over a 14-d recovery period. Overall, exposure to 50 000 µatm pCO2 had more pronounced physiological consequences compared with 15 000 µatm pCO2 . Over the first 7 d of exposure to 50 000 µatm pCO2 , the mRNA abundance of chitin synthase (cs), calmodulin (cam), and calmodulin-like protein (calp) were significantly affected, suggesting that shell formation and integrity may be altered during pCO2 exposure. After the removal of the pCO2 treatment, mussels may compensate for the acid-base and ion disturbances experienced during pCO2 exposure, and transcript levels of some regulators of biomineralization (carbonic anhydrase [ca], cs, cam, calp) as well as ion regulation (na+ -k+ -ATPase [nka]) were modulated. Effects of elevated pCO2 on heat shock protein 70 (hsp70) were limited in the present study. Overall, adult L. siliquoidea appeared to regulate factors associated with the control of biomineralization and ion regulation during and/or after the removal of pCO2 exposure. Environ Toxicol Chem 2018;37:538-550. © 2017 SETAC.

Effects of fluorine-containing usnic acid and fungus Beauveria bassiana on the survival and immune-physiological reactions of Colorado potato beetle larvae.

BACKGROUND: The search for compounds that interact synergistically with entomopathogenic fungi is aimed at enhancing the efficacy and stability of biological products against pest insects, for example, against the Colorado potato beetle (CPB). We hypothesized that fluorine-containing derivatives of usnic acid (FUA) might be candidates for the development of multicomponent bio-insecticides. The aim of this study was to analyze the co-influence of FUA and Beauveria bassiana on the survival and immune-physiological reactions of CPB larvae. RESULTS: Synergy between FUA and B. bassiana was observed after treatment of second, third and fourth larvae instars under laboratory conditions. Furthermore, synergy was observed in field trials in continental climate conditions in southeastern Kazakhstan. In a field experiment, the median lethal time was shortened three-fold, and cumulative mortality for 15 days increased by 36% in the combined treatment compared with a fungal infection alone. FUA treatment delayed larval development, decreased the total hemocyte count, and increased both the phenoloxidase activity in integuments and the detoxification enzyme rate in hemolymph. A combined treatment with fungus and FUA led to increases in the aforementioned changes. CONCLUSION: Toxicosis caused by FUA provides a stable synergistic effect between FUA and B. bassiana. The combination can be promising for the development of highly efficient products against CPB. © 2017 Society of Chemical Industry.

Impact of cationic polystyrene nanoparticles (PS-NH2) on early embryo development of Mytilus galloprovincialis: Effects on shell formation.

The potential release of nanoparticles (NPs) into aquatic environments represents a growing concern for their possible impact on aquatic organisms. In this light, exposure studies during early life stages, which can be highly sensitive to environmental perturbations, would greatly help identifying potential adverse effects of NPs. Although in the marine bivalve Mytilus spp. the effects of different types of NPs have been widely investigated, little is known on the effects of NPs on the developing embryo. In M. galloprovincialis, emerging contaminants were shown to affect gene expression profiles during early embryo development (from trocophorae-24 hpf to D-veligers-48 hpf). In this work, the effects of amino-modified polystyrene NPs (PS-NH2) on mussel embryos were investigated. PS-NH2 affected the development of normal D-shaped larvae at 48 hpf (EC50 = 0.142 mg/L). Higher concentrations (5-20 mg/L) resulted in high embryotoxicity/developmental arrest. At concentrations ≅ EC50, PS-NH2 affected shell formation, as shown by optical and polarized light microscopy. In these conditions, transcription of 12 genes involved in different biological processes were evaluated. PS-NH2 induced dysregulation of transcription of genes involved in early shell formation (Chitin synthase, Carbonic anhydrase, Extrapallial Protein) at both 24 and 48 hpf. Decreased mRNA levels for ABC transporter p-glycoprotein-ABCB and Lysozyme were also observed at 48 hpf. SEM observations confirmed developmental toxicity at higher concentrations (5 mg/L). These data underline the sensitivity of Mytilus early embryos to PS-NH2 and support the hypothesis that calcifying larvae of marine species are particularly vulnerable to abiotic stressors, including exposure to selected types of NPs.

Effects of phenanthrene on early development of the Pacific oyster Crassostrea gigas (Thunberg, 1789).

Phenanthnere (PHE) is a polycyclic aromatic hydrocarbon continuously discarded in the marine environment and bioavailable to many aquatic species. Although studies about PHE toxicity have been documented for adult oysters, the effects on early developmental stages are poorly characterized in bivalves. In this study, the effects of PHE (0.02 and 2.0µg.L-1) were evaluated on the embryogenesis and larval development of Crassostrea gigas. Toxicity bioassays, growth and deformities assessment, analysis of shell calcium abundance and transcript levels of genes related to xenobiotic biotransformation (CYP2AU2, CYP30C1), immune system (Cg-Tal) and tissue growth and shell formation (Ferritin, Insulin-like, Cg-Try, Calmodulin and Nacrein) were assayed in D-shape larvae after 24h of PHE exposure. At the highest concentration (2.0µg.L-1), PHE decreased the frequency of normal development (19.7±2.9%) and shell size (53.5±2.8mm). Developmental deformities were mostly related to abnormal mantle and shell formation. Lower calcium levels in oyster shells exposed to PHE 2.0µg.L-1 were observed, suggesting effects on shell structure. At this same PHE concentration, CYP30C1, Cg-Tal, Cg-Tyr, Calmodulin were upregulated and CYP2AU2, Ferritin, Nacrein, and Insulin-Like were downregulated compared to control larvae. At the lowest PHE concentration (0.02µg.L-1), it was observed a minor decrease in normal larval development (89,6±6%) and the remaining parameters were not affected. This is the first study to provide evidences that exposure to PHE can affect early oyster development at the molecular and morphological levels, possibly threatening this bivalve species.

Effects of ocean acidification on the shells of four Mediterranean gastropod species near a CO2 seep.

Marine CO2 seeps allow the study of the long-term effects of elevated pCO2 (ocean acidification) on marine invertebrate biomineralization. We investigated the effects of ocean acidification on shell composition and structure in four ecologically important species of Mediterranean gastropods (two limpets, a top-shell snail, and a whelk). Individuals were sampled from three sites near a volcanic CO2 seep off Vulcano Island, Italy. The three sites represented ambient (8.15pH), moderate (8.03pH) and low (7.73pH) seawater mean pH. Shell mineralogy, microstructure, and mechanical strength were examined in all four species. We found that the calcite/aragonite ratio could vary and increased significantly with reduced pH in shells of one of the two limpet species. Moreover, each of the four gastropods displayed reductions in either inner shell toughness or elasticity at the Low pH site. These results suggest that near-future ocean acidification could alter shell biomineralization and structure in these common gastropods.