Morphological, histological and behavioural change in two species of marine bivalves in response to environmental stress

<p>Mass mortality events (MMEs) occur when a disproportionate part of a population dies in a single event. The frequency of MMEs is increasing globally. In the past, MMEs have been linked to starvation, changes in environmental conditions and disease outbreaks. However, it is often unclear what the underlying cause of these events are. In New Zealand several MMEs have occurred in the bivalve species Austrovenus stutchburyi (Wood 1828) and Paphies subtriangulata (Wood 1828) with little known about the cause. Both of these species are recreationally harvested for consumption in New Zealand and have cultural significance. In order to better understand MMEs in these species we must first gain a better understanding of stress expression. Bivalves have few observable features and it is difficult to classify them as healthy or stressed without investigating immune change which can be quite costly. Some research has looked into how different cell types change in response to pollutants but few studies have researched how cell types change in response to environmental conditions. The aim of this research was to find novel ways of assessing if shellfish were healthy or stressed. Little is known about how shellfish respond to environmental stressors and this is the first study to look at several novel stress expressions simultaneously, in New Zealand shellfish. Histological, morphological and behavioural responses were measured in both A. stutchburyi and P. subtriangulata after treatment with increased temperature, lowered salinity and increased fine sediment input for up to 5 weeks. Temperature stress was the main stressor for P. subtriangulata (85% of overall mortality occurred in the heat treatment), salinity was the main stressor for A. stutchburyi (46% of overall mortality occurred in the salinity treatment), and fine sediment stress did not seem to have an effect on either species in this study. Overall, A. stutchburyi were more robust to the treatments, but low mortality occurred in both species (≤8%). Mortality correlated with time of year and was believed to be related to spawning in P. subtriangulata (48% of overall mortality occurred from October-November). Both species had a single histological marker, in A. stutchburyi this was change in gill morphology, and in P. subtriangulata this was change in digestive gland morphology. Several individual morphological features were identified as potential stress markers in A. stutchburyi and P. subtriangulata. Additionally, when removed from aquaria P. subtriangulata had impeded foot retraction time in the salinity treatment. The differences in stress markers shows the diversity of reactions to stressors even within New Zealand bivalves. This study provides a useful baseline in investigating how P. subtriangulata and A. stutchburyi respond to environmental stress. The histological slides produced during this investigation are an invaluable resource that can be used in future studies and in comparisons with archived specimens from known MMEs. Knowing how to detect signs of stress in these bivalves will help to predict MMEs in the future and aid in implementing processes to combat these events.</p>

Morphological, histological and behavioural change in two species of marine bivalves in response to environmental stress

<p>Mass mortality events (MMEs) occur when a disproportionate part of a population dies in a single event. The frequency of MMEs is increasing globally. In the past, MMEs have been linked to starvation, changes in environmental conditions and disease outbreaks. However, it is often unclear what the underlying cause of these events are. In New Zealand several MMEs have occurred in the bivalve species Austrovenus stutchburyi (Wood 1828) and Paphies subtriangulata (Wood 1828) with little known about the cause. Both of these species are recreationally harvested for consumption in New Zealand and have cultural significance. In order to better understand MMEs in these species we must first gain a better understanding of stress expression. Bivalves have few observable features and it is difficult to classify them as healthy or stressed without investigating immune change which can be quite costly. Some research has looked into how different cell types change in response to pollutants but few studies have researched how cell types change in response to environmental conditions. The aim of this research was to find novel ways of assessing if shellfish were healthy or stressed. Little is known about how shellfish respond to environmental stressors and this is the first study to look at several novel stress expressions simultaneously, in New Zealand shellfish. Histological, morphological and behavioural responses were measured in both A. stutchburyi and P. subtriangulata after treatment with increased temperature, lowered salinity and increased fine sediment input for up to 5 weeks. Temperature stress was the main stressor for P. subtriangulata (85% of overall mortality occurred in the heat treatment), salinity was the main stressor for A. stutchburyi (46% of overall mortality occurred in the salinity treatment), and fine sediment stress did not seem to have an effect on either species in this study. Overall, A. stutchburyi were more robust to the treatments, but low mortality occurred in both species (≤8%). Mortality correlated with time of year and was believed to be related to spawning in P. subtriangulata (48% of overall mortality occurred from October-November). Both species had a single histological marker, in A. stutchburyi this was change in gill morphology, and in P. subtriangulata this was change in digestive gland morphology. Several individual morphological features were identified as potential stress markers in A. stutchburyi and P. subtriangulata. Additionally, when removed from aquaria P. subtriangulata had impeded foot retraction time in the salinity treatment. The differences in stress markers shows the diversity of reactions to stressors even within New Zealand bivalves. This study provides a useful baseline in investigating how P. subtriangulata and A. stutchburyi respond to environmental stress. The histological slides produced during this investigation are an invaluable resource that can be used in future studies and in comparisons with archived specimens from known MMEs. Knowing how to detect signs of stress in these bivalves will help to predict MMEs in the future and aid in implementing processes to combat these events.</p>

Functional role of betalains in Disphyma australe under salinty stress

<p>Foliar betalainic plants are commonly found in dry and exposed environments such as deserts and sandbanks. This marginal habitat has led many researchers to hypothesise that foliar betalains provide tolerance to abiotic stressors such as strong light, drought, salinity and low temperatures. Among these abiotic stressors, soil salinity is a major problem for agriculture affecting approximately 20% of the irrigated lands worldwide. Betacyanins may provide functional significance to plants under salt stress although this has not been unequivocally demonstrated. The purpose of this thesis is to add knowledge of the various roles of foliar betacyanins in plants under salt stress. For that, a series of experiments were performed on Disphyma australe, which is a betacyanic halophyte with two distinct colour morphs in vegetative shoots. In chapter two, I aimed to find the effect of salinity stress on betacyanin pigmentation in D. australe and it was hypothesised that betacyanic morphs are physiologically more tolerant to salinity stress than acyanic morphs. Within a coastal population of red and green morphs of D. australe, betacyanin pigmentation in red morphs was a direct result of high salt and high light exposure. Betacyanic morphs were physiologically more tolerant to salt stress as they showed greater maximum CO₂ assimilation rates, water use efficiencies, photochemical quantum yields and photochemical quenching than acyanic morphs. Contrary to this, the green morphs, although possessing the ability to synthesise betalains in flower petals, did not produce betalains in vegetative shoots in response to salt stress. Moreover, green morphs, in terms of leaf photosynthesis, performed poorly under salinity stress. In chapter three I further investigated the physiological benefit of betacyanin accumulation in D. australe. I postulated that betacyanin in the leaves of D. australe can protect the salt stressed chloroplasts from harmful excessive light by absorbing significant amount of radiation. To test this, a novel experimental approach was used; the key biosynthetic step for betacyanin synthesis was identified, which was deficient in vegetative shoots of the green morphs. By supplying the product of this enzymatic reaction, L-DOPA, betacyanin synthesis could be induced in the leaves of green morphs. This model system was used to compare the photoprotective responses of red vs. green leaves. The L-DOPA induced betacyanic leaves showed similar responses (such as smaller reductions and faster recoveries of PSII and less H₂O₂ production than in the green leaves) to naturally betacyanic leaves when exposed to high light and salinity. The differences in photoinhibition between red and green leaves were attributed to the light absorbing properties of betacyanins. L-DOPA treated and naturally red leaves showed lower photoinactivation than green leaves when exposed to white or green light, although not when exposed to monochromatic (red) light. In chapter four, I used a similar experimental model to that in the third chapter and showed that other than photoprotection, betacyanins in leaves may be involved in salt tolerance by enhancing toxic ion (such as Na⁺) sequestration in betacyanic epidermal cells, storing Na⁺ away from sensitive mesophyll tissue. The Na⁺ localization between red and green leaves was compared after salinity treatment by using a sodium binding stain (SBFI-AM) and Cryo-SEM analysis. L-DOPA treated and natural red leaves sequestered Na⁺ ions to the epidermal cell layer. In contrast, green leaves retained Na⁺ in the mesophyll tissue, which suggested that red leaves were better equipped to tolerate salt-specific effects. Therefore, betacyanic plants were more tolerant to applied salinity stress and showed relatively higher growth rates than green morphs. The findings of this thesis provide a significant contribution to our understanding of the role of betacyanins in plants under salinity stress. My data suggest that the multi-faceted properties of betacyanins (such as their photoprotective function, and their involvement in sequestration of toxic ions) clearly provide a benefit to plants under salinity stress.</p>

Functional role of betalains in Disphyma australe under salinty stress

<p>Foliar betalainic plants are commonly found in dry and exposed environments such as deserts and sandbanks. This marginal habitat has led many researchers to hypothesise that foliar betalains provide tolerance to abiotic stressors such as strong light, drought, salinity and low temperatures. Among these abiotic stressors, soil salinity is a major problem for agriculture affecting approximately 20% of the irrigated lands worldwide. Betacyanins may provide functional significance to plants under salt stress although this has not been unequivocally demonstrated. The purpose of this thesis is to add knowledge of the various roles of foliar betacyanins in plants under salt stress. For that, a series of experiments were performed on Disphyma australe, which is a betacyanic halophyte with two distinct colour morphs in vegetative shoots. In chapter two, I aimed to find the effect of salinity stress on betacyanin pigmentation in D. australe and it was hypothesised that betacyanic morphs are physiologically more tolerant to salinity stress than acyanic morphs. Within a coastal population of red and green morphs of D. australe, betacyanin pigmentation in red morphs was a direct result of high salt and high light exposure. Betacyanic morphs were physiologically more tolerant to salt stress as they showed greater maximum CO₂ assimilation rates, water use efficiencies, photochemical quantum yields and photochemical quenching than acyanic morphs. Contrary to this, the green morphs, although possessing the ability to synthesise betalains in flower petals, did not produce betalains in vegetative shoots in response to salt stress. Moreover, green morphs, in terms of leaf photosynthesis, performed poorly under salinity stress. In chapter three I further investigated the physiological benefit of betacyanin accumulation in D. australe. I postulated that betacyanin in the leaves of D. australe can protect the salt stressed chloroplasts from harmful excessive light by absorbing significant amount of radiation. To test this, a novel experimental approach was used; the key biosynthetic step for betacyanin synthesis was identified, which was deficient in vegetative shoots of the green morphs. By supplying the product of this enzymatic reaction, L-DOPA, betacyanin synthesis could be induced in the leaves of green morphs. This model system was used to compare the photoprotective responses of red vs. green leaves. The L-DOPA induced betacyanic leaves showed similar responses (such as smaller reductions and faster recoveries of PSII and less H₂O₂ production than in the green leaves) to naturally betacyanic leaves when exposed to high light and salinity. The differences in photoinhibition between red and green leaves were attributed to the light absorbing properties of betacyanins. L-DOPA treated and naturally red leaves showed lower photoinactivation than green leaves when exposed to white or green light, although not when exposed to monochromatic (red) light. In chapter four, I used a similar experimental model to that in the third chapter and showed that other than photoprotection, betacyanins in leaves may be involved in salt tolerance by enhancing toxic ion (such as Na⁺) sequestration in betacyanic epidermal cells, storing Na⁺ away from sensitive mesophyll tissue. The Na⁺ localization between red and green leaves was compared after salinity treatment by using a sodium binding stain (SBFI-AM) and Cryo-SEM analysis. L-DOPA treated and natural red leaves sequestered Na⁺ ions to the epidermal cell layer. In contrast, green leaves retained Na⁺ in the mesophyll tissue, which suggested that red leaves were better equipped to tolerate salt-specific effects. Therefore, betacyanic plants were more tolerant to applied salinity stress and showed relatively higher growth rates than green morphs. The findings of this thesis provide a significant contribution to our understanding of the role of betacyanins in plants under salinity stress. My data suggest that the multi-faceted properties of betacyanins (such as their photoprotective function, and their involvement in sequestration of toxic ions) clearly provide a benefit to plants under salinity stress.</p>

The Jacalin-Related Lectin HvHorcH Is Involved in the Physiological Response of Barley Roots to Salt Stress

Salt stress tolerance of crop plants is a trait with increasing value for future food production. In an attempt to identify proteins that participate in the salt stress response of barley, we have used a cDNA library from salt-stressed seedling roots of the relatively salt-stress-tolerant cv. Morex for the transfection of a salt-stress-sensitive yeast strain (Saccharomyces cerevisiae YSH818 Δhog1 mutant). From the retrieved cDNA sequences conferring salt tolerance to the yeast mutant, eleven contained the coding sequence of a jacalin-related lectin (JRL) that shows homology to the previously identified JRL horcolin from barley coleoptiles that we therefore named the gene HvHorcH. The detection of HvHorcH protein in root extracellular fluid suggests a secretion under stress conditions. Furthermore, HvHorcH exhibited specificity towards mannose. Protein abundance of HvHorcH in roots of salt-sensitive or salt-tolerant barley cultivars were not trait-specific to salinity treatment, but protein levels increased in response to the treatment, particularly in the root tip. Expression of HvHorcH in Arabidopsis thaliana root tips increased salt tolerance. Hence, we conclude that this protein is involved in the adaptation of plants to salinity.

Effects of short-term salinity exposure on haemolymph osmolality, gill morphology and Na+/K+ - ATPase activity in Solenaia oleivora

In order to explore the physiological reaction to hyperosmotic environment, Solenaia oleivora were exposed to 2.23‰ salinity. In 48h, the hemolymph osmolality kept increasing, and the hemolymph protein concentration increased in the first 6h and then decreased significantly, while the free amino acid content increased in the first 24h and then kept stable (P < 0.05). The activity of Na+/K+-ATPase at 0h was significantly higher than other times in most organs except intestine, which was highest at 3h (P < 0.05). The ions concentration were also influenced. The concentration of Na+ rose in haemolymph, axe foot and intestine, but decreased in gill and hepatopancreas. In hemolymph, gill, hepatopancreases and adductor muscle, the K+ concentration was the highest at 0h, while in axe foot and intestine, it showed a positive tendency. The concentration of Cl- in haemolymph, adductor muscle, intestine and axe foot were positively correlated with treatment time, while hepatopancreas showed opposite tendency. High salinity stress caused a difference in the gill histological structure, the gill structure shrunk, the gill lamellas space and shrinking degree showed an enlarging trend with salinity treatment time.

Ion-specific Limitations of Sodium Chloride and Calcium Chloride on Growth, Nutrient Uptake, and Mycorrhizal Colonization in Northern and Southern Highbush Blueberry

Excess salinity is becoming a prevalent problem for production of highbush blueberry (Vaccinium L. section Cyanococcus Gray), but information on how and when it affects the plants is needed. Two experiments, including one on the northern highbush (Vaccinium corymbosum L.) cultivar, Bluecrop, and another on the southern highbush (V. corymbosum interspecific hybrid) cultivar, Springhigh, were conducted to investigate their response to salinity and assess whether any suppression in growth was ion specific or due primarily to osmotic stress. In both cases, the plants were grown in soilless media (calcined clay) and fertigated using a complete nutrient solution containing four levels of salinity [none (control), low (0.7–1.3 mmol·d−1), medium (1.4–3.4 mmol·d−1), and high (2.8–6.7 mmol·d−1)] from either NaCl or CaCl2. Drainage was minimized in each treatment except for periodic determination of electrical conductivity (EC) using the pour-through method, which, depending on the experiment, reached levels as high as 3.2 to 6.3 dS·m−1 with NaCl and 7.8 to 9.5 dS·m−1 with CaCl2. Total dry weight of the plants was negatively correlated to EC and, depending on source and duration of the salinity treatment, decreased linearly at a rate of 1.6 to 7.4 g·dS−1·m−1 in ‘Bluecrop’ and 0.4 to 12.5 g·dS−1·m−1 in ‘Springhigh’. Reductions in total dry weight were initially similar between the two salinity sources; however, by the end of the study, which occurred at 125 days in ‘Bluecrop’ and at 111 days in ‘Springhigh’, dry weight declined more so with NaCl than with CaCl2 in each part of the plant, including in the leaves, stems, and roots. The percentage of root length colonized by mycorrhizal fungi also declined with increasing levels of salinity in Bluecrop and was lower in both cultivars when the plants were treated with NaCl than with CaCl2. However, leaf damage, which included tip burn and marginal necrosis, was greater with CaCl2 than with NaCl. In general, CaCl2 had no effect on uptake or concentration of Na in the plant tissues, whereas NaCl reduced Ca uptake in both cultivars and reduced the concentration of Ca in the leaves and stems of Bluecrop and in each part of the plant in Springhigh. Salinity from NaCl also resulted in higher concentrations of Cl and lower concentrations of K in the plant tissues than CaCl2 in both cultivars. The concentration of other nutrients in the plants, including N, P, Mg, S, B, Cu, Fe, Mn, and Zn, was also affected by salinity, but in most cases, the response was similar between the two salts. These results point to ion-specific effects of different salts on the plants and indicate that source is an important consideration when managing salinity in highbush blueberry.

Exogenous Spermine Mitigate Adversities of Salinity Induced Oxidative Stress through Antioxidant Metabolites in Wheat

Change in climatic scenarios due to global warming is characterized by extreme climate variability, land and water degradation which resulted in water scarcity. Accumulation of salts at the surface and sub-surface layers of soils affect crop production of major cereals which is a constraint in sustainable food production. Salinity is a major challenge to tackle wheat cultivation and harness productivity in arid and semi-arid regions of India. In the present investigation, mitigation of salinity induced oxidative stress through exogenous application of spermine (Spm) in four wheat genotypes was studied in relation to antioxidant metabolites. The levels of O2.- increased with increasing levels of salinity in wheat flag leaves. DBW 88 showed the levels of O2.- of 11.75 nmol g-1 FW and 15.74 nmol g-1 FW (at 8 dSm-1 and 12 dSm-1 respectively) at 21 Days After Sowing (DAS) and application of Spm decreased the O2.- content under control and saline stressed conditions at 8 dSm-1 and 12 dSm-1. Hydrogen peroxide content was increased with increasing levels of salinity in all the wheat varieties at 21 DAS. However, the increase was more in the case of DBW 88 when compared with HD 3086. Treatment of Spm decreased the H2O2 content when compared with control and saline stressed wheat varieties. The malondialdehyde (MDA) content was increased with increasing levels of salinity at 21 DAS. The highest increase in MDA content was seen in DBW 88 whereas the lowest increase was found in Kharchia 65. Application of Spm decreased the MDA content under control at both levels of salinity treated wheat varieties. The carotenoid content decreased with increasing levels of salinity in all four wheat varieties. However, the decrease was more in DBW 88 when compared with other varieties viz. HD 3086, Kharchia 65 and KRL 210 at 21 DAS. Exogenous Spm increased the carotenoids content in all four wheat varieties irrespective of the salinity. The leaves of Kharchia 65 and KRL 210 had higher levels of ascorbic acid as compared to that of DBW 88 and HD 3086. Increased content of carotenoid was observed in Spm-treated wheat. Exogenous application of Spm increased the ascorbic acid content in control at both levels of salt stress. The glutathione content increased with an increase in salinity treatment in all the varieties however, a higher increase was observed in Kharchia 65. Exogenous Spm increased the glutathione content in all the varieties irrespective of salinity stress. The results presented in the study indicated that the exogenous application of Spm improved their tolerance levels under salinity.

Effect of NaCl or Macronutrient-Imposed Salinity on Basil Crop Yield and Water Use Efficiency

Cascade hydroponics, that is, the application of the circular economy concept in greenhouse hydroponic crops, may be considered as an alternative means to increase water and nutrient use efficiency in greenhouses. In such systems, the drained nutrient solution from a crop may be used as input in a second crop. However, the second (secondary) crop in the loop must be a crop that is less sensitive to salinity than the first (primary) crop. In the present study, the salinity tolerance of basil plants grown in rockwool and nutrient film technique (NFT) systems was investigated in order to study the potential of using a basil crop as a secondary crop in a cascade hydroponic system. In total, 4 electrical conductivity (EC) levels of the irrigation nutrient solution were tested (2, 4, 6, and 8 dS m−1), and salinity was imposed by NaCl or by macronutrients. Plant growth varied across the different substrates, with those grown in the NFT system being less affected as opposed to the rockwool-grown basil plants, which showed a significant growth decrease with EC values higher than 4 dS m−1. This relatively low growth pattern was associated with a decrease in water use efficiency (WUE) in the rockwool system. On the contrary, in the NFT system, the continuous flow of the nutrient solution in the root zone of the plants contributed to the alleviation of negative salinity effects, yielding up to 30 kg FM m−2 WUE even for the plants irrigated with the highest salinity treatment (8 dS m−1). The majority of macro- and micronutrients in the leaf tissue of basil were positively affected by the higher levels of conductivity in the nutrient solution. Therefore, basil cultivation could be efficiently incorporated as a secondary crop in a cascade NFT cropping system. This would contribute to drainage management in hydroponics, as the crop could be irrigated through the moderately saline drainage from a primary crop due to either NaCl or high nutrient accumulation in the leachates.