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Marine bivalves are exposed to several environmental stressors depending on fluctuations in abiotic factors (pollutants, salinity, and temperature) and biotic factors, including ascidian biofouling. Increased seawater temperature may lead to oxidative stress in marine animals, whereas the additional burden presented by ascidian biofouling narrows their thermal window and can cause a sharper decline in shellfish health, with subsequent effects on shellfish aquaculture. The aim of the present study was to investigate the role of seawater temperature variations in the severity of the stress caused by ascidian biofouling to Mytilus galloprovincialis cell machinery, particularly in the antioxidant defense system, and the apoptotic and autophagic pathways. Fouled and nonfouled mussels were seasonally collected, and several key indicators of the previously mentioned cellular processes were determined in their mantle and posterior adductor muscle tissues. Results indicate that ascidian biofouling increases hypoxic conditions and oxidative stress, subsequently triggering cell death pathways in mussels. Although the stress caused by ascidians may be the consequence of both reduced feeding and oxygen consumption, it affects the mussels to a greater extent than seasonality. The induced stress by biofouling, along with elevated water temperatures in summer, may severely harm mussel physiology and fitness, adversely affecting aquaculture sustainability. [ABSTRACT FROM AUTHOR]

Mussel farming in the Vistonikos Gulf (North Aegean Sea) constitutes an activity of high socioeconomic importance. The wider Vistonikos Gulf area consists of three basins (Porto Lagos Lagoon, Vistonikos Bay, and Vistonida Lake) rich in organic material. Oceanographic features, sea currents, meteorological and climatic conditions, and primary productivity of the wider Vistonikos Gulf marine area favour bivalve farming establishment. However, secondary factors associated with climate change may negatively affect these farming establishments. In the present study, historical oceanographic and meteorological data associated with the bivalve's biology and culture is reviewed. The Vistonikos Gulf demonstrates a suitable area for mussels and other bivalves' maintenance and aquaculture development. However, occasional restrictions imposed to mussel farm units in the past are related to meteorological extremes. Thus, monitoring seawater physicochemical properties within farming units may prevent harmful effects (e.g., mortality, heat and oxidative stress) through the establishment of an early warning system indicating the translocation or harvest of the reared bivalves.

A 2-yr sand fly (Diptera: Psychodidae) seasonality study was performed in Attica Region, Greece, from June 2017 until November 2018, aiming also to detect the presence of Leishmaniainfection in the collected sand flies. In total, 701 sand flies were collected from urban areas within the Attica Region using BG-Sentinel traps, set weekly in eight fixed sites. Five species were identified morphologically and molecularly, namely Phlebotomus tobbi(Adler and Theodor), which was most the most commonly collected species, followed by P. Neglectus(Tonnoir), P. papatasi(Scopoli), P. simici(Theodor), and Sergentomyia minuta(Rondani). During both survey years sand fly populations peaked in late August to early September. Fifty-nine monospecific pools were examined for Leishmaniadetection by analyzing the ITS1 nuclear region using both RFLPs and sequencing, seven of which were found positive. LeishmaniaDNA was identified as L. infantumin six pools (five P. papatasiand one P. tobbi), whereas in one P. papatasipool LeishmaniaDNA was identified as L. tropica.This is the first time that L. tropicahas been detected in naturally infected sand flies from the Attica Region as well as in central Greece, while previously it has only been detected in sand flies collected from Central Macedonia (Northern Greece).

Sheep’s reproductive physiology in temperate latitudes (such as Greece), is characterized by seasonality and is also regulated by photoperiodic exposure. Melatonin is the key hormone involved in this regulation. However, the melatonin secretion and therefore the ewes reproductive activity underlies variation, proposed to be linked with the melatonin receptor subtype 1A (MNTR1A) gene structure. This study was designed to investigate the polymorphism of the MNTR1Agene in a local Greek sheep breed and to determine its potential association with reproductive seasonality. Two groups of farmed ewes, each consisted of 30 individuals, were chosen. Males were introduced in both groups in spring (April). The first group consisted of ewes that showed reproductive activity in spring (May), while the second of ewes that showed reproductive activity 3 months later, in summer. The PCR–RFLP methodology was carried out on a 824-bp DNA fragment of the MTNR1Aexon 2 using the RsaI restriction endonuclease. The electrophoretic procedure revealed three genotypes, C/C, C/Tand T/T. Specifically, 44 animals showed the C/Cgenotype (28 from the first group and 16 from the second), 14 the C/Tgenotype (2 from the first and 12 from the second) and 2 animals had the T/Tgenotype (both from the second group). Statistical analysis indicated a positive correlation between genotype and reproductive seasonality, with C/Cgenotype playing a crucial role in out-of-season reproduction activity.

Organisms can modify and increase their thermal tolerance faster and more efficiently after a brief exposure to sublethal thermal stress. This response is called ‘heat hardening’ as it leads to the generation of phenotypes with increased heat tolerance. The aim of this study was to investigate the impact of heat hardening on the metabolomic profile of Mytilus galloprovincialis in order to identify the associated adjustments of biochemical pathways that might benefit the mussels' thermal tolerance. Thus, mussels were exposed sequentially to two different phases (heat hardening and acclimation phases). To gain further insight into the possible mechanisms underlying the metabolic response of the heat-hardened M. galloprovincialis, metabolomics analysis was complemented by the estimation of mRNA expression of phosphoenolpyruvate carboxykinase (PEPCK), pyruvate kinase (PK) and alternative oxidase (AOX) implicated in the metabolic pathways of gluconeogenesis, glycolysis and redox homeostasis, respectively. Heat-hardened mussels showed evidence of higher activity of the tricarboxylic acid (TCA) cycle and diversification of upregulated metabolic pathways, possibly as a mechanism to increase ATP production and extend survival under heat stress. Moreover, formate and taurine accumulation provide an antioxidant and cytoprotective role in mussels during hypoxia and thermal stress. Overall, the metabolic responses in non-heat-hardened and heat-hardened mussels underline the upper thermal limits of M. galloprovincialis, set at 26°C, and are in accordance with the OCLTT concept. The ability of heat-hardened mussels to undergo a rapid gain and slow loss of heat tolerance may be an advantageous strategy for coping with intermittent and often extreme temperatures.

Background: Microcystins are emerging marine biotoxins, produced by potentially toxic cyanobacteria. Their presence has been reported in aquatic animals in Greek freshwater, while data are few in marine environments. Since the climate change induces eutrophication and harmful algal blooms in coastal marine ecosystems affecting the public health, further research on microcystins’ presence in marine waters is required. The aim of this study was to examine the potential presence of microcystins in mussels Mytilus galloprovincialisin the largest farming areas in Thermaikos gulf, in Northern Greece, and to investigate their temporal and spatial distribution, adding to the knowledge of microcystins presence in Greek Mediterranean mussels. Results: A 4-year microcystins’ assessment was conducted from 2013 to 2016, in farmed Mediterranean mussels M. galloprovincialis, in five sampling areas in Thermaikos gulf, in northern Greece, where the 90% of the Greek mussels’ farming activities is located. The isolation of potentially toxic cyanobacteria was confirmed by molecular methods. An initial screening was performed with a qualitative and quantitative direct monoclonal (DM) ELISA and results above 1 ng g−1were confirmed for the occurrence of the most common microcystins-RR, -LR and -YR, by Ultra High Performance Liquid Chromatography (UHPLC) coupled with a high- resolution mass spectrometer (HRMS) (Orbitrap analyzer). Microcystin-RR and microcystin-LR were detected, while the intensity of microcystin-YR was below the method detection limit. Most samples that exhibited concentrations above 1 ng g-1were detected during the warm seasons of the year and especially in spring. Results indicated an overestimation of the ELISA method, since concentrations ranged between 0.70 ± 0.15 ng g−1and 53.90 ± 3.18 ng g−1, while the confirmation denoted that the levels of microcystins were 6 to 22 times lower. Conclusions: Microcystin-RR and microcystin-LR were detected for the first time in mussel M. galloprovincialis, harvested from farms in Thermaikos gulf, in Central Macedonia, Greece. Their presence was linked to potentially toxic cyanobacteria. Bioaccumulation was observed in digestive gland, while the concentrations in muscles were found extremely low. Samples with levels above 1 ng g−1were observed mostly during spring, confirming the seasonal distribution of microcystins. The comparison of the results by the ELISA and the LC-Orbitrap MS method indicated an overestimation of concentration by the ELISA method.

Nondestructive techniques allow the isolation of genomic DNA, without damaging the morphological features of the specimens. Though such techniques are available for numerous insect groups, they have not been applied to any member of the medically important families of mosquitoes (Diptera: Culicidae) and phlebotomine sand flies (Diptera: Psychodidae). This study presents Mild-Vectolysis, the first nondestructive DNA extraction methodology for vouchering taxa of mosquitoes and sand flies, which provided sufficient amounts of DNA, tested in a verified barcode (cytochrome oxidase I gene), while preserving their morphological integrity. Application of the method to sand flies allowed successful insect identification post DNA extraction, as all basic taxonomical structures necessary for identification (pharynx, cybarium, and genitalia) remained intact. The development of the methodology was more challenging in mosquitoes, due to the fragility of key morphological characters (scales and color). A small modification of the lysis buffer concentration, in combination with the adjustment of the incubation time, a postlysis freezing stage, and the avoidance of ethanol, achieved the extraction of sufficient DNA quantity, while preserving the integument of the mosquitoes, although a small proportion of the scales and the color still appeared to have been lost. In addition to the practicality and efficiency of our methodology, preserving of the original insect specimen post DNA extraction is highly advantageous, as it allows for 1) utilization of the specimen for further analysis and 2) storage for vouchering.

AbstractTwelve putative-resistant (R) redroot pigweed populations were collected in sunflower and soybean fields located in northeastern Greece, after repeated exposure to the acetolactate synthase (ALS)–inhibiting herbicides imazamox and tribenuron-methyl. Studies were conducted to determine the resistance status to these two ALS-inhibiting herbicides and evaluate alternative postemergence and preemergence herbicides for effective control. Two susceptible (S) populations were also included for comparison. Among the 12 putative-R populations studied in the whole-plant dose–response pot experiments, 11 were characterized as cross-resistant (R) to the imidazolinone imazamox and the sulfonylurea tribenuron-methyl. In contrast, the putative R5 and the two reference populations (S1, S2) populations were found to be susceptible. Sequencing of the ALSgene revealed that a point mutation (TGG to TTG at position 574) was selected in domain B, where in combination with domain A the majority of point mutations conferring resistance have been detected, resulting in an amino acid substitution from tryptophan (Trp) to leucine (Leu) in the 11 R populations. By contrast, all sequenced plants of the three susceptible populations were found with the wild-type allele encoding Trp574. The labeled rate of the postemergence herbicides tembotrione and dicamba provided fair to excellent control of the populations with ALS cross-resistance. In contrast, at this rate the preemergence herbicides S-metolachlor + terbuthylazine, isoxaflutole, aclonifen, metribuzin, and pendimethalin provided excellent control. These findings strongly suggest that 11 redroot pigweed populations have evolved cross-resistance to ALS-inhibiting herbicides, but viable options for chemical control of this weed still exist.