The uncoupling of respiration in plant mitochondria: keeping reactive oxygen and nitrogen species under control.

Plant mitochondrial respiration involves the operation of various alternative pathways. These pathways participate, both directly and indirectly, in the maintenance of mitochondrial functions though they do not contribute to energy production, being uncoupled from the generation of an electrochemical gradient across the mitochondrial membrane and thus from ATP production. Recent findings suggest that uncoupled respiration is involved in reactive oxygen species (ROS) and nitric oxide (NO) scavenging, regulation, and homeostasis. Here we discuss specific roles and possible functions of uncoupled mitochondrial respiration in ROS and NO metabolism. The mechanisms of expression and regulation of the NDA-, NDB- and NDC-type non-coupled NADH and NADPH dehydrogenases, the alternative oxidase (AOX), and the uncoupling protein (UCP) are examined in relation to their involvement in the establishment of the stable far-from-equilibrium state of plant metabolism. The role of uncoupled respiration in controlling the levels of ROS and NO as well as inducing signaling events is considered. Secondary functions of uncoupled respiration include its role in protection from stress factors and roles in biosynthesis and catabolism. It is concluded that uncoupled mitochondrial respiration plays an important role in providing rapid adaptation of plants to changing environmental factors via regulation of ROS and NO.

Method for detection of mtDNA damages for evaluating of pesticides toxicity for bumblebees (Bombus terrestris L.).

Bumblebees are important for crop pollination. Currently, the number of pollinators is decreasing worldwide, which is attributed mostly to the widespread use of pesticides. The aim of this work was to develop a method for assessing the genotoxicity of pesticides for the Bombus terrestris L. bumblebee using long-range PCR of mitochondrial DNA fragments. We have developed a panel of primers and assessed the genotoxicity of the following pesticides: imidacloprid, rotenone, deltamethrin, difenocanozole, malathion, metribuzin, penconazole, esfenvalerate, and dithianon. All pesticides (except imidacloprid) inhibited mitochondrial respiration fueled by pyruvate + malate; the strongest effect was observed for rotenone and difenocanozole. Three pesticides (dithianon, rotenone, and difenocanozole) affected the rate of H2O2 production. To study the pesticide-induced DNA damage in vitro and in vivo, we used three different mtDNA. The mtDNA damage was observed for all studied pesticides. Most of the studied pesticides caused significant damage to mtDNA in vitro and in vivo when ingested. Our results indicate that all tested pesticides, including herbicides and fungicides, can have a toxic effect on pollinators. However, the extent of pesticide-induced mtDNA damage in the flight muscles was significantly less upon the contact compared to the oral administration.

Cyclic Synthetic Peroxides Inhibit Growth of Entomopathogenic Fungus Ascosphaera apis without Toxic Effect on Bumblebees.

In recent years, the number of pollinators in the world has significantly decreased. A possible reason for this is the toxic effects of agrochemicals reducing the immunity of insects that leads to their increased susceptibility to pathogens. Ascosphaera apis is a dangerous entomopathogenic fungus, afflicting both honeybees and bumblebees. We investigated fungicide activity of cyclic synthetic peroxides against A. apis isolated from Bombus terrestris L. The peroxides exhibited high mycelium growth inhibition of A. apis up to 94-100% at concentration 30 mg/L. EC50 values were determined for the most active peroxides. Two peroxides showed higher antifungal activity against A. apis than the commercial fungicide Triadimefon. The studied peroxides did not reduce the ability of bumblebees to fly and did not lead to the death of bumblebees. A new field of application for peroxides was disclosed.

Unique features of flight muscles mitochondria of honey bees (Apis mellifera L.).

Honey bees Apis mellifera L. are one of the most studied insect species due to their economic importance. The interest in studying honey bees chiefly stems from the recent rapid decrease in their world population, which has become a problem of food security. Nevertheless, there are no systemic studies on the properties of the mitochondria of honey bee flight muscles. We conducted a research of the mitochondria of the flight muscles of A. mellifera L. The influence of various organic substrates on mitochondrial respiration in the presence or absence of adenosine diphosphate (ADP) was investigated. We demonstrated that pyruvate is the optimal substrate for the coupled respiration. A combination of pyruvate and glutamate is required for the maximal respiration rate. We also show that succinate oxidation does not support the oxidative phosphorylation and the generation of membrane potential. We also studied the production of reactive oxygen species by isolated mitochondria. The greatest production of H2 O2 (as a percentage of the rate of oxygen consumed) in the absence of ADP was observed during the respiration supported by α-glycerophosphate, malate, and a combination of malate with another NAD-linked substrate. We showed that honey bee flight muscle mitochondria are unable to uptake Ca2+ -ions. We also show that bee mitochondria are able to oxidize the respiration substrates effectively at the temperature of 50°Ð¡ compared to Bombus terrestris mitochondria, which were more adapted to lower temperatures.

Evaluation of the toxicity of fungicides to flight muscle mitochondria of bumblebee (Bombus terrestris L.).

Insects pollinate 75% of crops used for human consumption. Over the last decade, a substantial reduction in the abundance of pollinating insects has been recorded and recognized as a severe matter for food supply security. Many of the important food crops destined for human consumption are grown in greenhouses. A unique feature of greenhouse agriculture is the extensive use of fungicides to curb multiple fungal infections. The most widely used pollinating insects in greenhouses are commercially reared bumblebees. However, there is no data regarding the toxicity of fungicides to bumblebee mitochondria. To fill this gap in knowledge, we examined the effects of 16 widely used fungicides on the energetics of the flight muscles mitochondria of Bombus terrestris. We found that diniconazole and fludioxonil uncoupled the respiration of mitochondria; dithianon and difenoconazole inhibited it. By analyzing the action of these inhibitors on mitochondrial respiration and generation of reactive oxygen species, we concluded that difenoconazole inhibited electron transport at the level of Complex I and glycerol-3-phosphate dehydrogenase. Dithianon strongly inhibited succinate dehydrogenase and glycerol-3-phosphate dehydrogenase. It also strongly inhibited mitochondrial oxidation of NAD-linked substrates or glycerol 3-phosphate, but it had no effect on the enzymatic activity of Complex I. It may be suggested that dithianon inhibits electron transport downstream of Complex I, likely at multiply sites.

Female reproductive tract microbiome and early miscarriages.

Miscarriage is one of the main causes of reproductive loss, which can lead to a number of physical and psychological complications and other long-term consequences. However, the role of vaginal and uterine microbiome in such complications is poorly understood. To review the published data on the function of the female reproductive tract microbiome in the pathogenesis of early miscarriages. The articles published over the past 20 years and deposited in PubMed, Google Academy, Scopus, Elibrary, ResearchGate, and EBSCO databases were analyzed. The review presents new data on the impact of the vaginal and uterine microbiome on the local immunity, including defense against sexually transmitted infections, and its association with other factors of miscarriages. The studies on the microbiome of non-pregnant women with recurrent miscarriages in the anamnesis, patients undergoing IVF, and pregnant women with miscarriages, as well as new directions in the microbiome research are discussed. The majority of studies have demonstrated that the dominant species of the vaginal and uterine microbiome in patients with early miscarriages are non-Lactobacillus bacteria. As many of these bacteria have not previously been detected by cultural studies and their role in obstetric complications is not well defined, further research on the female reproductive tract microbiome, including the microbiome of the cervix uteri, is needed to develop new approaches for the prognosis and prevention of miscarriages.

Changes in the Microbiome Profile in Different Parts of the Intestine in Piglets with Diarrhea.

Determining the taxonomic composition of microbial consortia of the piglet intestine is of great importance for pig production. However, knowledge on the variety of the intestinal microbiome in newborn piglets is limited. Piglet diarrhea is a serious gastrointestinal disease with a high morbidity and mortality that causes great economic damage to the pig industry. In this study, we investigated the microbiome of various sections of the piglet intestine and compared the microbiome composition of healthy and diarrheal piglets using high-throughput sequencing of the 16S rRNA gene. The results showed that bacteria of the Lactobacillus genus were the most common in the ileum, while Fusobacterium and Bacteroides dominated in the rectum. Comparing the microbiome composition of healthy and diarrheal piglets revealed a reduced number of Lactobacillus bacteria as a hallmark of diarrhea, as did an increased content of representatives of the Escherichia-Shigella genus and a reduced number of Bacteroides, which indicates the contribution of these bacteria to the development of diarrhea in piglets. The relative abundance of Enterococcus bacteria was higher in the diarrhea group. Although some bacteria of this genus are commensals, a small number of species may be associated with the development of diarrhea in piglets. Therefore, our results indicate that the gut microbiome may be an important factor in the development of diarrhea in piglets.

Microbiota of Cow's Milk with Udder Pathologies.

Mastitis is the most common disease for cattle, causing great economic losses for the global dairy industry. Recent studies indicate the multi-agent and microbiome diversity of this disease. To understand the nature of mastitis and investigate the role of the microbiome in the development of pathologies in the udder of bovines, we performed NGS sequencing of the 16S rRNA gene of cow's milk with pathologies of the udder. The obtained data show a significant increase in the Cutibacterium, Blautia, Clostridium sensu stricto 2, Staphylococcus, Streptococcus and Microbacterium genera for groups of cows with udder pathologies. Increasing relative abundance of the Staphylococcus and Streptococcus genera was associated with subclinical mastitis. Our data show that a relative increase in abundance of the Staphylococcus and Microbacterium genera may be an early sign of infection. We have shown, for the first time, an increase in the Colidextribacter, Paeniclostridium and Turicibacter genera in groups of cows with mastitis. These results expand our understanding of the role of the microbiome in the development of bovine mastitis.

High-Throughput 16S rRNA Gene Sequencing of Butter Microbiota Reveals a Variety of Opportunistic Pathogens.

Microbial contamination of dairy products with a high fat content (e.g., butter) has been studied insufficiently. No studies using modern molecular methods to investigate microbial communities in butter have been conducted so far. In this work, we used high-throughput sequencing and Sanger sequencing of individual bacterial colonies to analyze microbial content of commercially available butter brands. A total of 21 samples of commercially available butter brands were analyzed. We identified a total of 94 amplicon sequence variants corresponding to different microbial taxa. The most abundant lactic acid bacteria in butter were Lactobacillus kefiri, Lactobacillus parakefiri, Lactococcus taiwanensis and Lactococcus raffinolactis. A large amount of Streptococcus spp. bacteria (87.9% of all identified bacteria) was found in one of the butter samples. Opportunistic pathogens such as Bacillus cereus group, Pseudomonas aeruginosa, Cronobacter spp., Escherichia coli, Listeria innocua, Citrobacter spp., Enterococcus spp., Klebsiella pneumonia were detected. The analyzed butter samples were most strongly contaminated with bacteria from the Bacillus cereus group, and to a lesser extent - with Cronobacter spp. and Enterococcus spp. The plating and Sanger sequencing of individual colonies revealed the presence of Enterobacter cloacae and Staphylococcus epidermidis. The Sanger sequencing also showed the presence of Cronobacter sakazakii in butter which can be dangerous for children under the age of 1 year. We demonstrated that butter is a good growth medium for opportunistic pathogenic bacteria. Our data indicate that despite the fact that butter is a dairy product with a long shelf life, it should be subjected to quality control for the presence of opportunistic bacteria.

A Molecular Method for the Identification of Honey Bee Subspecies Used by Beekeepers in Russia.

Apis mellifera L. includes several recognized subspecies that differ in their biological properties and agricultural characteristics. Distinguishing between honey bee subspecies is complicated. We analyzed the Folmer region of the COX1 gene in honey bee subspecies cultivated at bee farms in Russia and identified subspecies-specific SNPs. DNA analysis revealed two clearly distinct haplogroups in A. melliferamellifera. The first one was characterized by multiple cytosine-thymine (thymine-cytosine) transitions, one adenine-guanine substitution, and one thymine-adenine substitution. The nucleotide sequence of the second haplogroup coincided with sequences from other subspecies, except the unique C/A SNP at position 421 of the 658-bp Folmer region. A. melliferacarnica and A. melliferacarpatica could be distinguished from A. melliferamellifera and A. melliferacaucasica by the presence of the A/G SNP at position 99 of the 658-bp Folmer region. The G/A SNP at position 448 was typical for A. melliferacarnica. A. melliferacaucasicaCOX1 sequence lacked all the above-mentioned sites. We developed a procedure for rapid identification of honey bee subspecies by PCR with restriction fragment length polymorphism (RFLP) using mutagenic primers. The developed molecular method for honey bee subspecies identification is fast and inexpensive.

DNA barcoding and morphological analysis for rapid identification of most economically important crop-infesting Sunn pests belonging to Eurygaster Laporte, 1833 (Hemiptera, Scutelleridae).

The genus Eurygaster Laporte, 1833 includes ten species five of which inhabit the European part of Russia. The harmful species of the genus is E. integriceps. Eurygaster species identification based on the morphological traits is very difficult, while that of the species at the egg or larval stages is extremely difficult or impossible. Eurygaster integriceps, E. maura, and E. testudinaria differ only slightly between each other morphologically, E. maura and E. testudinaria being almost indiscernible. DNA barcoding based on COI sequences have shown that E. integriceps differs significantly from these closely related species, which enables its rapid and accurate identification. Based on COI nucleotide sequences, three species of Sunn pests, E. maura, E. testudinarius, E. dilaticollis, could not be differentiated from each other through DNA barcoding. The difference in the DNA sequences between the COI gene of E. integriceps and COI genes of E. maura and E. testudinarius was more than 4%. In the present study DNA barcoding of two Eurygaster species was performed for the first time on E. integriceps, the most dangerous pest in the genus, and E. dilaticollis that only inhabits natural ecosystems. The PCR-RFLP method was developed in this work for the rapid identification of E. integriceps.