The Status of Marine Mussel Pollution Research in South Africa (2012-2022).

The growing human population requires more food each year, and seafood products can help meet this demand if clean water resources are available for their growth. Farmed and wild mussels are environmentally friendly seafood with many health benefits to human consumers, but they can also pose a health risk if they are harvested from areas where marine anthropogenic pollution is uncontrolled or unmonitored. While the coastline in South Africa has long been assumed to be pristine, a growing number of recent studies are raising contamination concerns. Baseline studies establish a wide range of anthropogenic pollutants to be present in the marine environment, specifically in urbanised or industrialised areas like major cities or harbours. This review summarises how mussels could pose health risks to human consumers and the current research that is being conducted by private researchers and institutions in South Africa. The review emphasises the need for more research in the field and for governmental pollution monitoring data to be released to the public.

Acid-base adjustments and first evidence of denticle corrosion caused by ocean acidification conditions in a demersal shark species.

Global ocean acidification is expected to chronically lower the pH to 7.3 (>2200 µatm seawater pCO2) by the year 2300. Acute hypercapnia already occurs along the South African west and south coasts due to upwelling- and low-oxygen events, with increasing frequency. In the present project we investigated the impact of hypercapnia on the endemic demersal shark species Haploblepharus edwardsii. Specifically, we experimentally analysed acid-base regulation during acute and chronic hypercapnia, the effects of chronic hypercapnia on growth rates and on denticle structure- and composition. While H. edwardsii are physiologically well adapted to acute and chronic hypercapnia, we observed, for the first time, denticle corrosion as a result of chronic exposure. We conclude that denticle corrosion could increase denticle turnover and compromise hydrodynamics and skin protection.

Effects of chronic hypercapnia and elevated temperature on the immune response of the spiny lobster, Jasus lalandii.

The West Coast rock lobster (WCRL), Jasus lalandii, inhabits highly variable environments frequented by upwelling events, episodes of hypercapnia and large temperature variations. Coupled with the predicted threat of ocean acidification and temperature change for the coming centuries, the immune response in this crustacean will most likely be affected. We therefore tested the hypothesis that chronic exposure to hypercapnia and elevated seawater temperature will alter immune function of the WCRL. The chronic effects of four combinations of two stressors (seawater pCO2 and temperature) on the total number of circulating haemocytes (THC) as well as on the lobsters' ability to clear (inactivate) an injected dose of Vibrio anguillarum from haemolymph circulation were assessed. Juvenile lobsters were held in normocapnic (pH 8.01) or hypercapnic (pH 7.34) conditions at two temperatures (15.6 and 18.9 °C) for 48 weeks (n = 30 lobster per treatment), after which a subsample of lobsters (n = 8/treatment), all at a similar moult stage, were selected from each treatment for the immune challenge. Baseline levels of haemocytes (THC ml-1) and bacteria (CFU ml-1) in their haemolymph were quantified 24 h prior to bacterial challenge. Lobsters were then challenged by injecting 4 × 104V. anguillarum per g body weight directly into the cardiac region of each lobster and circulating haemocyte and culturable bacteria were measured at 20 min post challenge. No significant differences in THC ml-1 (p < 0.05) were observed between any of the treatment groups prior to the bacterial challenge. However lobsters chronically exposed to a combination of hypercapnia and low temperature had significantly higher (p < 0.05) THCs post-challenge in comparison with lobsters chronically exposed to hypercapnia and high temperature. A significant interactive effect was recorded between temperature and pH for the post-challenge THC data (two-way ANOVA, p = 0.0025). Lobster were very efficient at rendering an injected dose of bacteria non-culturable, with more than 83% of the theoretical challenge dose (∼1.7 × 105Vibrio ml-1 haemolymph) inactivated within the first 10 min following injection. Although differences in the inactivation of V. anguillarum were observed between treatment groups, none of these differences were significant. Clearance efficiency was in the following order: Hypercapnia/low temperature > normocapnia/high temperature > normocapnia/low temperature > hypercapnia/high temperature. This study demonstrated that despite chronic exposure to combinations of reduced seawater pH and high temperature, the WCRL was still capable of rapidly rendering an injected dose of bacteria non-culturable.

The winter pack-ice zone provides a sheltered but food-poor habitat for larval Antarctic krill.

A dominant Antarctic ecological paradigm suggests that winter sea ice is generally the main feeding ground for krill larvae. Observations from our winter cruise to the southwest Atlantic sector of the Southern Ocean contradict this view and present the first evidence that the pack-ice zone is a food-poor habitat for larval development. In contrast, the more open marginal ice zone provides a more favourable food environment for high larval krill growth rates. We found that complex under-ice habitats are, however, vital for larval krill when water column productivity is limited by light, by providing structures that offer protection from predators and to collect organic material released from the ice. The larvae feed on this sparse ice-associated food during the day. After sunset, they migrate into the water below the ice (upper 20 m) and drift away from the ice areas where they have previously fed. Model analyses indicate that this behaviour increases both food uptake in a patchy food environment and the likelihood of overwinter transport to areas where feeding conditions are more favourable in spring.

Acid-base balance and changes in haemolymph properties of the South African rock lobsters, Jasus lalandii, a palinurid decapod, during chronic hypercapnia.

Few studies exist reporting on long-term exposure of crustaceans to hypercapnia. We exposed juvenile South African rock lobsters, Jasus lalandii, to hypercapnic conditions of pH 7.3 for 28 weeks and subsequently analysed changes in the extracellular fluid (haemolymph). Results revealed, for the first time, adjustments in the haemolymph of a palinurid crustacean during chronic hypercapnic exposure: 1) acid-base balance was adjusted and sustained by increased bicarbonate and 2) quantity and oxygen binding properties of haemocyanin changed. Compared with lobsters kept under normocapnic conditions (pH 8.0), during prolonged hypercapnia, juvenile lobsters increased bicarbonate buffering of haemolymph. This is necessary to provide optimum pH conditions for oxygen binding of haemocyanin and functioning of respiration in the presence of a strong Bohr Effect. Furthermore, modification of the intrinsic structure of the haemocyanin molecule, and not the presence of molecular modulators, seems to improve oxygen affinity under conditions of elevated pCO2.

Physiological and morphological colour change in Antarctic krill, Euphausia superba: a field study in the Lazarev Sea.

Antarctic krill, Euphausia superba, is very susceptible to harmful solar radiation because of its unique genetic setup. Exposure occurs in spring to autumn during vertical diel migration and during occasional daytime surface-swarming. We have investigated colour change in Antarctic krill, Euphausia superba, during summer and winter in the Lazarev Sea in response to ultraviolet radiation (UVR) and photosynthetically active radiation (PAR). Short-term physiological colour change and long-term (seasonal) morphological colour change are present. Both are facilitated by a single type of monochromatic red chromatophore, i.e. erythrophores, of 20-450 microm diameter. Superficial erythrophores cover large dorsal areas, especially above vital organs (brain, sinus glands), additional 'profound' erythrophores cover internal organs (heart, gut, nerve cords). Short-term change in light regime causes rapid physiological colour change along dense bundles of microtubules: pigment disperses into chromorhizae upon exposure to PAR and UVA and to a lesser extent to UVB. Darkness leads to aggregation of pigment in the centre and hence blanching. There is no circadian rhythm in the dispersal state of erythrophores present in winter. Physiological colour change in adult krill is two to three times more rapid in summer than in winter. Furthermore, seasonal changes in light regime also result in a profound morphological colour change: in summer animals, abdominal astaxanthin concentration is 450% and erythrophore count is 250-480% higher than in winter krill. We conclude from our results, that pigmentation of E. superba serves in the protection from harmful solar radiation and is adapted to the varying diel and seasonal light conditions.

Flight fuel and neuropeptidergic control of fuel mobilisation in the twig wilter, Holopterna alata (Hemiptera, Coreidae).

The corpus cardiacum of the twig wilter Holopterna alata contains a factor that elicits increases in the concentration of lipids in the haemolymph of twig wilters and migratory locusts and causes hypertrehalosaemia in American cockroaches. A hyperlipaemic neuropeptide was isolated from corpora cardiaca of H. alata in a single high-performance liquid chromatography step. The primary sequence of this peptide was assigned by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, biological assay and co-elution with the synthetic peptide. The adipokinetic peptide of H. alata is an octapeptide with the sequence pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp amide denoted Schgr-AKH-II which was sequenced previously from the corpora cardiaca of a number of Caelifera, Ensifera and some Hymenoptera. A dose of 1pmol of synthetic Schgr-AKH-II causes a pronounced hyperlipaemic effect in the twig wilter. Physiological experiments with the twig wilter reveal that during flight periods of 3 min, the normally low carbohydrate concentration in the haemolymph is significantly diminished, whereas the lipid concentration stays constant in most cases. During a subsequent rest period of 60 min after a 3 min flight episode, however, the concentration of lipids in the haemolymph increases substantially and significantly, indicating that lipids, too, are a major fuel during flight of twig wilters. This is corroborated by the activation of the enzyme triacylglycerol (TAG) lipase in the fat body, but not in the flight muscles, by injection of 5 pmol of synthetic Schgr-AKH-II, the endogenous adipokinetic hormone that is thought to be released during flight. Moreover, in the thorax there is a significant decrease in the concentration of glycogen and lipids measured after flight plus 60 min of rest compared to non-flown twig wilters, whereas no significant changes were monitored for these substrates stored in the abdomen. When the change in lipid class composition was analysed during flight plus 60 min of rest, TAG which comprised the major class in all compartments analysed (thorax, abdomen, haemolymph) was significantly reduced in abdomen and thorax, and diacylglycerol was significantly increased in all three compartments. From all the data collected, it is concluded that lipids are the major fuel class for flight in H. alata and that the contribution of carbohydrates is minimal.

Endocrine control of TAG lipase in the fat body of the migratory locust, Locusta migratoria.

Aspects of the role and activation of the enzyme triacylglycerol lipase (TAG lipase) in the fat body of the migratory locust Locusta migratoria were investigated. TAG lipase is under the hormonal control of the three endogenous adipokinetic peptides of the migratory locust, Locmi-AKH-I, Locmi-AKH-II and Locmi-AKH-III. Injection of low doses (5-10 pmol) of each peptide causes an increase in lipase activity. The activation of lipase is time dependent: an elevated activity was recorded 15 min after injection of 10 pmol Locmi-AKH-I and maximum activation was reached after 45-60 min. The activation of TAG lipase is also dose-dependent. Doses of 2 pmol of each Locmi-AKH had no effect, whereas 5 pmol caused a significant activation. Maximum activation is reached with a dose of 10 pmol. Analogues of the second messengers cAMP (cpt-cAMP) and IP(3) (F-IP(3)) both activate the enzyme glycogen phosphorylase whereas only cpt-cAMP, but not F-IP(3), activates TAG lipase; cpt-cAMP elevates the lipid levels in the haemolymph. Activation of lipase is specific to the three endogenous AKH peptides: 5 pmol of the endogenous peptide Locmi-HrTH and 10 pmol of corazonin failed to activate lipase. High doses of octopamine did not activate lipase nor did they elevate the lipid concentration in the haemolymph. TAG lipase is stimulated by flight activity but activation is slower than that of glycogen phosphorylase: after 30 min of flight or after 5 min of flight plus 1h of subsequent rest, activity of TAG lipase is increased, but not immediately after 5 min of flight. In contrast, glycogen phosphorylase is activated significantly after 5 min of flight. These activation patterns of the two enzymes mirror-image the concentration of their substrates in the haemolymph: there is a significant decrease in the concentration of carbohydrates after 5 min of flight, whereas no change of the concentration of lipids can be measured after such short time of flight activity; however, a subsequent rest period of 1h is sufficient to increase the lipid concentration.

Unique translational modification of an invertebrate neuropeptide: a phosphorylated member of the adipokinetic hormone peptide family.

Separation of an extract of corpora cardiaca from the protea beetle, Trichostetha fascicularis, by single-step RP (reverse-phase)-HPLC and monitoring of tryptophan fluorescence resulted in two distinctive peaks, the material of which mobilized proline and carbohydrates in a bioassay performed using the beetle. Material from one of these peaks was; however, inactive in the classical bioassays of locusts and cockroaches that are used for detecting peptides belonging to the AKH (adipokinetic hormone) family. After enzymatically deblocking the N-terminal pyroglutamic acid (pGlu) residue in the peptide material and sequencing by Edman degradation, a partial sequence was obtained: (pGlu)-Ile-Asn-Met-Thr-Xaa-Gly-Trp. The complete sequence was deduced from ESI-MS(n) (electrospray ionization multi-stage-MS); position six was identified as a phosphothreonine residue and the C-terminus is amidated. The peptide, code-named Trifa-CC, was chemically synthesized and used in confirmatory experiments to show that the primary structure had been correctly assigned. To our knowledge, this is the first report of a phosphorylated invertebrate neuropeptide. Synthetic Trifa-CC co-elutes with the natural peptide, found in the gland of the protea beetle, after RP-HPLC. Moreover, the natural peptide can be dephosphorylated by alkaline phosphatase and the product of that reaction has the same retention time as a synthetic nonphosphorylated octapeptide which has the same sequence as Trifa-CC. Finally, synthetic Trifa-CC has hypertrehalosaemic and hyperprolinaemic biological activity in the protea beetle, but even high concentrations of synthetic Trifa-CC are inactive in locusts and cockroaches. Hence, the correct peptide structure has been assigned. Trifa-CC of the protea beetle is an unusual member of the AKH family that is unique in its post-translational modification. Since it increases the concentration of carbohydrates and proline in the haemolymph when injected into the protea beetle, and since these substrates are also used during flight, we hypothesize that Trifa-CC controls the mobilization of these metabolites in the protea beetle.

Activation of triacylglycerol lipase in the fat body of a beetle by adipokinetic hormone.

The activation of triacylglycerol lipase and the stimulation of proline synthesis in the fat body of the fruit beetle Pachnoda sinuata by the endogenous octapeptide hormone Melme-CC (pQLNYSPDWa), which belongs to the family of insect adipokinetic hormones, were studied, and the correlation of both events investigated. At rest, the activity of triacylglycerol lipase in the fat body of the beetle was higher than in the fat body of the American cockroach, Periplaneta americana, but lower than in the migratory locust, Locusta migratoria. Triacylglycerol lipase of the beetle is activated by: (a) injection of synthetic Melme-CC and (b) the stimulus of flight. Activation of lipase by Melme-CC is time-dependent. Injection of cpt-cAMP activates triacylglycerol lipase in the fat body and causes an increase in the concentration of proline in the haemolymph at the expense of alanine. In contrast, injection of F-inositol-1,4,5-phosphate does not affect the activation state of lipase, nor the levels of amino acids in the haemolymph. High doses of octopamine do not activate lipase. Furthermore, activity of fat body lipase and proline concentration in the haemolymph both follow a circadian rhythm: both parameters are high in the morning, whereas they are low in the evening. When transfer of Melme-CC, released from the corpora cardiaca, to the thorax/abdomen is prevented by neck-ligation, the activity of lipase, as well as the circulating proline levels are low. Regression analysis revealed that activity of triacylglycerol lipase is positively correlated to proline concentration in the haemolymph, whereas there is a negative correlation of the enzyme activity and alanine level in the haemolymph. From these results we conclude that the activation of fat body triacylglycerol lipase by Melme-CC in P. sinuata stimulates proline synthesis. Proline is one of the major substrates to power flight activity in the beetle.

Red pigment-concentrating hormone is not limited to crustaceans.

A peptide that was previously assumed to occur exclusively in crustaceans is found in the corpora cardiaca of the stinkbug, Nezara viridula. The sequence of the peptide was deduced from the multiple MS(N) electrospray mass data as that of an octapeptide: pGlu-Ile/Leu-Asn-Phe-Ser-Pro-Gly-Trp amide. This peptide with Leu at position 2 is known as crustacean red pigment-concentrating hormone and code-named Panbo-RPCH. The ambiguity about the amino acid at position 2, Leu or Ile, was solved by isolating the peptide in a single-step by reversed-phase HPLC and establishing co-elution with authentic Panbo-RPCH but not with the Ile(2)-analog. When injected into stinkbugs, synthetic Panbo-RPCH elicited an increase of lipids in the haemolymph. Thus, it is assumed that Panbo-RPCH functions in the stinkbug as a lipid-mobilizing hormone.

Mode of action of neuropeptides from the adipokinetic hormone family.

Neuropeptides of the adipokinetic hormone (AKH) family regulate inter alia mobilisation of various substrates from stores in the fat body of insects during episodes of flight. How is this achieved? In insects which exclusively oxidise carbohydrates for flight (cockroaches), or which oxidise carbohydrates in conjunction with lipids (locusts) or proline (a number of beetles), the endogenous AKHs bind to a G(q)-protein-coupled receptor, activate a phospholipase C and the resulting inositol trisphosphate releases Ca(2+) from internal stores. In addition, influx of extracellular Ca(2+) is increased and, via a kinase cascade, glycogen phosphorylase is activated, glucose-1-phosphate produced, and transformed to trehalose, which is released into the haemolymph. In locusts, additionally, adenylate cyclase is activated and cyclic AMP is synthesised. In insects which use lipids for sustained flight (locust, tobacco hornworm moth) or proline for flight (certain beetles), adenylate cyclase is activated after the AKHs bind to their respective G(s)-protein-coupled receptor. The resulting cyclic AMP, together with the messengers intra- and extracellular Ca(2+), activate a triacylglycerol lipase, which results in the production of 1,2 diacylglycerols (in locusts, moths) or (hypothetically) free fatty acids (fruit beetle).

The role of Ins(1,4,5)P(3) in signal transduction of the metabolic neuropeptide Mem-CC in the cetoniid beetle, Pachnoda sinuata.

We have investigated the role of inositol triphosphate, Ins(1,4,5)P(3), in the transduction of the hypertrehalosaemic and hyperprolinaemic signal of the endogenous neuropeptide Mem-CC in the cetoniid beetle Pachnoda sinuata. Flight and injection of Mem-CC into the haemocoel of the beetle induce an increase of Ins(1,4,5)P(3) levels in the fat body of the beetle. When Mem-CC is co-injected with U 73122, which is an inhibitor of phospholipase C, this effect is abolished. Mem-CC also elevates Ins(1,4,5)P(3) concentration in fat body pieces in vitro. The increase in Ins(1,4,5)P(3) levels is tissue-specific and does not occur in brain and flight muscles. Elevation of the Ins(1,4,5)P(3) levels upon injection of Mem-CC is time- and dose-dependent: the maximum response is reached after 3 min and a dose of 10 pmol is needed. Compounds that mimic the action of cAMP (cpt-cAMP, forskolin) do not influence the concentration of Ins(1,4,5)P(3), while those that stimulate G-proteins (aluminium fluoride and cholera toxin) cause an increase of Ins(1,4,5)P(3) levels. The application (in vivo and in vitro) of F-Ins(1,4,5)P(3), an Ins(1,4,5)P(3) analogue that penetrates the cell membrane, causes a mobilisation of carbohydrate reserves via the activation of glycogen phosphorylase but does not stimulate proline synthesis. In addition, U 73122 abolishes the hypertrehalosaemic but not the hyperprolinaemic effect of Mem-CC. The results suggest that the hypertrehalosaemic signal of Mem-CC is mediated via an increase of Ins(1,4,5)P(3) levels in the fat body of P. sinuata.

Beetles' choice--proline for energy output: control by AKHs.

Many beetle species use proline and carbohydrates in a varying ratio to power flight. The degree of contribution of either fuel varies widely between species. In contrast, dung beetle species investigated, thus far, do not have any carbohydrate reserves and rely completely on proline to power energy-costly activities such as flight and, probably, walking and ball-rolling. While the fruit beetle, Pachnoda sinuata, uses proline and carbohydrates equally during flight, proline is solely oxidised during endothermic pre-flight warm-up, as well as during flight after prolonged starvation. Thus, proline seems to be the essential fuel for activity in beetles, even in flightless ones and in those that use proline in combination with carbohydrates; the latter can be completely substituted by proline in certain circumstances. It is apparent from the rapid decline of energy substrates in flight muscles and haemolymph after the onset of flight that mobilisation of stored fuels of the fat body is necessary for prolonged flight periods. This task is performed by AKH-type neuropeptides. In beetles, like in other insects, these peptides mobilise glycogen via activation of glycogen phosphorylase. They also stimulate proline synthesis from alanine and acetyl-CoA in the fat body. Acetyl-CoA is derived from the beta-oxidation of fatty acids and we propose that the neuropeptides activate triacylglycerol lipase.

Physiological and biochemical aspects of flight metabolism in cocoon-enclosed adults of the fruit beetle, Pachnoda sinuata.

We studied several aspects of flight metabolism in cocoon-enclosed adults of the fruit beetle Pachnoda to investigate their flight capability. The majority of adults which were forcefully removed from their pupal cocoon flew off within 5 min of exposure to bright sunlight. Most of the beetles which did not fly voluntarily were, however, capable of flight. Compared with 2-4 week old adults of the same species, cocoon-enclosed adults have higher reserves of glycogen in flight muscles and fat body, whereas the level of total carbohydrates in the haemolymph and the concentration of proline in haemolymph, flight muscles and fat body were similar.Enzymes involved in carbohydrate breakdown (MDH, GAPDH) were more active in flight muscles and fat body of cocoon-enclosed adults compared with adults, while enzymes of proline metabolism in the flight muscles (AlaT, NAD-ME) and fat body (AlaT, NADP-ME) had similar activities in cocoon-enclosed adults and adults. An enzyme of the beta-oxidation of fatty acids (HOAD) had similar activities in flight muscles and fat body of cocoon-enclosed adults and adults.Mitochondria isolated from flight muscles of adults removed prematurely from their cocoon favour the oxidation of proline and pyruvate. Pyruvate, however, is oxidized at higher rates than by mitochondria isolated from flight muscles of adults.During a short lift-generating flight, cocoon-enclosed adults proved that their flight muscles are capable of strong flight performance. During these flights, cocoon-enclosed adults consume proline and carbohydrates at a similar rate to that of adults.The endogenous AKH peptide, Mem-CC, has hyperprolinaemic and hypertrehalosaemic activity in cocoon-enclosed adults. The hypertrehalosaemic effect, however, is stronger in cocoon-enclosed adults than in adults.The content of Mem-CC in corpora cardiaca of larvae (3rd instar), cocoon-enclosed adults and 1 day-old adults is similar at 5-6 pmol per pair of corpora cardiaca, whereas it is higher in 10 day-old adults and 20 day-old adults (37 and 15 pmol per pair corpora cardiaca, respectively).From these results we conclude that cocoon-enclosed adults comply with all the prerequisites for flight performance before they leave their pupal cocoon. Furthermore, cocoon-enclosed adults have a more pronounced carbohydrate-based metabolism before they leave their cocoon compared with adults, which suggests that carbohydrate breakdown is mainly involved in such activities as leaving the cocoon and burrowing activity thereafter.