High-resolution (mtDNA) melting analysis for simple and efficient characterization of Africanized honey bee Apis mellifera (Hymenoptera:Apidae).

Analysis of the mtDNA variation in Apis mellifera L. has allowed distinguishing subspecies and evolutionary lineages by means of different molecular methods; from RFLP, to PCR-RFLP and direct sequencing. Likewise, geometric morphometrics (GM) has been used to distinguish Africanized honey bees with a high degree of consistency with studies using molecular information. High-resolution fusion analysis (HRM) allows one to quickly identify sequence polymorphisms by comparing DNA melting curves in short amplicons generated by real-time PCR (qPCR). The objective of this work was to implement the HRM technique in the diagnosis of Africanization of colonies of A. mellifera from Argentina, using GM as a validation method. DNA was extracted from 60 A. mellifera colonies for mitotype identification. Samples were initially analyzed by HRM, through qPCRs of two regions (485 bp/385 bp) of the mitochondrial cytochrome b gene (cytb). This technique was then optimizing to amplify a smaller PCR product (207 bp) for the HRM diagnosis for the Africanization of colonies. Of the 60 colony samples analyzed, 41 were classified as colonies of European origin whereas 19 revealed African origin. All the samples classified by HRM were correctly validated by GM, demonstrating that this technique could be implemented for a rapid identification of African mitotypes in Apis mellifera samples.

Chronic bee paralysis virus (CBPV) in South American non-Apis bees.

Chronic bee paralysis virus (CBPV) is a positive single-stranded RNA virus that exhibits a worldwide distribution. Although the effects of this virus on honeybees' health are well known, its presence in other bee species has not been fully studied. In this work, CBPV was detected in several native bees from Argentina, including Bombus pauloensis, Halictillus amplilobus, Peponapis fervens, and members of the genus Xylocopa. Here, we report for the first time the presence of CBPV in native bees from South America.

Endometrial expression of key genes related to fertility in repeat breeder and non-repeat breeder cows.

The aim of the present study was to compare the endometrial gene expression of epidermal growth factor receptor (EGFR), nodal growth differentiation factor (NODAL), prostaglandin-endoperoxide synthase 2 (PTGS2), oestrogen receptor 1 (ESR1) and progesterone receptor (PGR) in repeat breeder cows (RBC) and non-RBC during diestrus. Endometrial samples were collected by cytobrush technique and stored in RNA stabilizing solution at -20°C until RT-qPCR analysis. Differences in endometrial mRNA expression of selected genes were assessed by ANOVA and simple (r) and the partial correlations (rp) among selected genes were performed. Results demonstrated that mRNA expression of EGFR and NODAL were higher in RBC than in non-RBC (3 and 25-fold change, p < .01 and p < .01, respectively), while the mRNA expression of PTGS2 was lower (1.56-fold change, p < .01). Although there were no differences detected in the mRNA expression of ESR1 and PGR, there was a positive correlation between the expression of ESR1 and EGFR (0.84, p < .05) and a negative correlation between PGR and PTGS2 (-0.49, p < .05). In conclusion, the difference on the endometrial mRNA expression of the genes included in the study between RBC and non-RBC indicates a deregulation of important mechanisms that are vital to establish a successful pregnancy. Thus, the present study provides useful insight as a base for future studies to elucidate the causes of RBC.

On the characterization of NaDEHP/n-heptane nonaqueous reverse micelles: the effect of the polar solvent.

The behavior of two polar solvents, ethylene glycol (EG) and dimethylformamide (DMF), entrapped in sodium bis-(2-ethylhexyl) phosphate (NaDEHP)/n-heptane reverse micelles (RMs) was investigated using dynamic light scattering (DLS), molecular probe absorption and FT-IR spectroscopy. DLS results reveal the formation of RMs containing EG and DMF as a polar component. To the best of our knowledge this is the first report where both polar solvents are entrapped by the NaDEHP surfactant to effectively create RMs. We use the solvatochromism behavior of the molecular probe, 1-methyl-8-oxyquinolinum betaine (QB), and FT-IR spectroscopy to investigate the physicochemical properties of the non-aqueous RMs. Our results demonstrate that the NaDEHP surfactant interacts through hydrogen bonds with EG at the EG/NaDEHP interface and this interaction is responsible for destroying the bulk structure of pure solvent EG when entrapped in NaDEHP RMs. On the other hand, when DMF is incorporated inside the RMs the bulk structure of DMF is destroyed upon encapsulation by the Na-DMF interaction at the DMF/NaDEHP interface. Our results are completely different than the one observed for DMF/n-heptane/AOT. Our results show how the physicochemical properties, such as micropolarity, microviscosity and hydrogen bond interaction, of nonaqueous NaDEHP/n-heptane RMs interfaces can be dramatically changed by simply using different non-aqueous polar solvents. Thus, these results can be very useful to employ these novel RMs as nanoreactors since the dimensions of the RMs are around 10 to 20 nm.

Overexpression of the insulin-like growth factor 1 receptor (IGF-1R) is associated with malignancy in familial pheochromocytomas and paragangliomas.

CONTEXT: Pheochromocytomas and paragangliomas (pheo/pgl) are neuroendocrine tumours derived from chromaffin cells. Although mostly benign, up to 26% of pheo/pgl will undergo malignant transformation. Reliable histological signs to differentiate benign pheo/pgl from malignant tumours are currently lacking. Increased IGF-1R expression has been shown during progression to metastatic phenotypes of several types of cancer. OBJECTIVE: To analyse the distribution and expression of the IGF-1R in pheo/pgl of different genetic origin and degree of malignancy. MEASUREMENTS: We studied the expression of the IGF-1R protein by immunohistochemistry, in 40 primary tumours from patients with pheo/pgl from different genetic aetiology (11 of 29 metastatic/nonmetastatic diseases). RESULTS: We found a strong association between increased expression of IGF-1R and malignant behaviour regardless of the age at diagnosis and the genetic aetiology. IGF-1R labelling was mostly weak in primary tumours from patients with nonmetastatic pheo/pgl. Conversely, intense IGF-1R labelling was predominant in cases of pheo/pgl with confirmed metastatic disease. The risk of metastases was 11·7 times higher if tumour IGF-1R labelling was intense independently of age at diagnosis. The probability of remaining free of metastases was higher in patients with pheo/pgl scored weak for IGF-1R at 60 months and more than twofold higher at 120 months of follow-up than in patients with intense IGF-1R labelling in their primary tumours. CONCLUSIONS: Our results strongly suggest that IGF-1R is associated with malignancy in familial pheo/pgl and that IGF-1R expression in the primary tumour might be a useful tool to detect those patients harbouring pheo/pgl who have an increased risk of metastasis.

Gastrointestinal parasites of domestic sheep from Patagonia throughout historical times: A paleoparasitological approach.

Sheep husbandry in Patagonia, Argentina, started at the beginning of the 20th century from European settlers. Sanitary management is minimal, which has serious implications for the sheep health. Sheep can be infested by diverse parasites, with well over 150 species of internal and external parasites reported worldwide. Gastrointestinal parasitism is one of the most common and important infections in sheep concerning the health status, and is the cause of significant morbidity and mortality, which generates considerable production losses. The present work is the first paleoparasitological study of sheep coprolites from Patagonia throughout historical times. Fifty-seven coprolites from the 'Cueva Peligro' archaeological site (Patagonia, Argentina) were analyzed using the Lutz spontaneous sedimentation technique and the modified Faust flotation technique. Ancient DNA (aDNA) study was carried out in order to identify the zoological origin of coprolites. The results obtained from Cytb analysis confirmed the identity of the host as Ovis aries (domestic sheep). A total of 39 coprolites examined were positive for parasites by at least one of the studied methods. Thirty eight samples were positive by spontaneous sedimentation and 10 samples were positive by the modified Faust technique. The parasitic fauna found was Trichuris sp. (Trichinellida: Trichuridae), Nematodirus sp., Nematodirus spathiger (Strongylida, Trichostrongyloidea), two unidentified Strongylida-type egg morphotypes, Fasciola hepatica (Trematoda: Digenea) and coccidia oocysts of Eimeria spp. (Apicomplexa: Eimeriidae). The modified Faust technique provided satisfactory results in terms of sensitivity for the detection of Eimeria spp. The use of this methodology as a routine procedure enables the processing of ancient samples, in order to increase recoveries. These results show the importance of integrating different diagnostic approaches in order to optimize parasitic findings. The recorded parasite diversity appears to have not changed over the last 120 years. The study displayed the presence of different parasitic species which suggests potential exposure to parasitic diseases through the historical times, both for sheep and for other domestic and wild mammals from Patagonia. Also, suggests the presence of fasciolosis, a zoonotic disease that implies a potential risk for Patagonian populations in the past.

Detection of Apicystis bombi (Apicomplexa: Neogregarinorida) in carpenter bees of Argentina.

Historically, the neogregarine Apicystis bombi was isolated almost exclusively from bumble bees (Bombus spp.) where it disrupts adipose tissue, increasing hosts' mortality rates. Records in solitary bees are scarce worldwide. To check for its presence in carpenter bees (genus Xylocopa), campaigns were performed in Argentina capturing 154 individuals of five species (X. augusti, X. splendidula, X. atamisquensis, X. frontalis, X. nigrocincta). The presence of A. bombi was detected by molecular means in X. augusti, X. atamisquensis, and X. nigrocincta in four of the nine provinces screened. The pathogenesis and eventual impact that A. bombi may cause in individuals or populations of Xylocopa species remain unknown. The presence of A. bombi in northern Argentina would be contradictory to the hypothesis that its occurrence is the exclusive result of its introduction to South America through invasive, infected exotic bumble bees.

The gut microbiome of solitary bees is mainly affected by pathogen assemblage and partially by land use.

Pollinators, including solitary bees, are drastically declining worldwide. Among the factors contributing to this decline, bee pathogens and different land uses are of relevance. The link between the gut microbiome composition and host health has been recently studied for social pollinators (e.g. honeybees), whereas the information related to solitary bees is sparse. This work aimed at the characterization of the gut microbiome of the solitary bees Xylocopa augusti, Eucera fervens and Lasioglossum and attempted to correlate the gut microbial composition with the presence and load of different pathogens and land uses. Solitary bees were sampled in different sites (i.e. a farm, a natural reserve, and an urban plant nursery) showing different land uses. DNA was extracted from the gut, 16S rRNA gene amplified and sequenced. Eight pathogens, known for spillover from managed bees to wild ones, were quantified with qPCR. The results showed that the core microbiome profile of the three solitary bees significantly varied in the different species. Pseudomonas was found as the major core taxa in all solitary bees analyzed, whereas Lactobacillus, Spiroplasma and Sodalis were the second most abundant taxa in X. augusti, E. fervens and Lasioglossum, respectively. The main pathogens detected with qPCR were Nosema ceranae, Nosema bombi and Crithidia bombi, although differently abundant in the different bee species and sampling sites. Most microbial taxa did not show any correlation with the land use, apart from Snodgrassella and Nocardioides, showing higher abundances on less anthropized sites. Conversely, the pathogens species and load strongly affected the gut microbial composition, with Bifidobacterium, Apibacter, Serratia, Snodgrassella and Sodalis abundance that positively or negatively correlated with the detected pathogens load. Therefore, pathogens presence and load appear to be the main factor shaping the gut microbiome of solitary bees in Argentina.

Transplacental infection by bovine alphaherpesvirus type 1 induces protein expression of COX-2, iNOS and inflammatory cytokines in fetal lungs and placentas.

Bovine alphaherpesvirus type 1 (BoAHV-1) is associated with respiratory and reproductive syndromes. Until present the immunologic mechanisms involved in BoAHV-1 abortion are partially known. We studied key elements of the innate immune response in the placentas and fetal lungs from cattle experimentally-inoculated with BoAHV-1. These tissues were analyzed by histopathology. Furthermore, virus identification was performed by qPCR and the expression of the inflammatory cytokines such as tumor necrosis factor-alpha, interleukin 1-alpha and inflammatory mediators like inducible nitric oxide synthase and cyclooxeganse-2 was evaluated by immunohistochemistry. The viral transplacental infection was confirmed by the detection of BoAHV-1 by qPCR in the placenta and fetal organs, which revealed mild inflammatory lesions. Inducible nitric oxide synthase immunolabelling was high in the lungs of infected fetuses and placentas, as well as for tumor necrosis factor-alpha in the pulmonary parenchyma and cyclooxeganse-2 in fetal annexes. However, the expression of interleukin 1-alpha was weak in these organs. To our knowledge, this is the first study that provides strong evidence of an early immune response to BoAHV-1 infection in the conceptus. Advances in the knowledge of the complex immunological interactions at the feto-maternal unit during BoAHV-1 infection are needed to clarify the pathogenesis of abortion.

Comparison between two anionic reverse micelle interfaces: the role of water-surfactant interactions in interfacial properties.

The water/sodium bis(2-ethylhexyl) phosphate (NaDEHP) reverse micelle (RM) system is revisited by using, for the first time, molecular probes to investigate interface properties. The solvatochromic behavior of 1-methyl-8-oxyquinolinium betaine (QB) and 6-propionyl-2-(N,N-dimethyl)aminonaphthalene (PRODAN) in the water/NaDEHP/toluene system is studied, and the results are compared with those obtained in water/sodium 1,4-bis(2-ethylhexyl) sulfosuccinate (AOT)/toluene RM media. The results demonstrate that the micropolarity, microviscosity, interfacial water structure, molecular probe partition, and intramolecular electron-transfer processes are dramatically altered for NaDEHP RM interfaces in comparison to the AOT systems. Because of organic nonpolar solvent penetration into the interface, NaDEHP RM media offer an interface with lower micropolarity and microviscosity than AOT media. Also, the interfacial water in the NaDEHP system shows enhanced water-water hydrogen-bond interaction in comparison with bulk water. The AOT RM interface represents a unique environment for PRODAN to undergo dual emission.

Origin and evolution of somatic cell testicular tumours in transgenic mice.

Transgenic mice bearing a construct in which the expression of the SV40 oncogene is directed by the AMH promoter (AT mice) develop testicular tumours in adult life. We aimed to study early steps of tumour development and characterize tumours at different ages by histological, morphometric, and immunohistochemical techniques. One- to 3-month-old AT mice depicted multifocal Leydig cell hyperplasia. The testicular volume occupied by interstitial tissue was significantly higher in 3-month-old AT mice in comparison with littermate controls. Between 5 1/2 and 7 months, microscopic interstitial tumours developed that progressively evolved to form large confluent areas of high mitotic index in 7- to 14-month-old AT mice. Tumour cells had the characteristics and histoarchitecture of Leydig cells, or formed solid cord-like structures reminiscent of those seen in Sertoli cell tumours. Hyperplastic areas and tumours diffusely expressed 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in Leydig cell areas. AMH expression was negative in Leydig cell conglomerates and tumours and variable in cord-like tumours. The SV40 T antigen and markers of cell proliferation (PCNA) were intensely positive in hyperplastic cells and tumours. Control mice of similar ages showed neither hyperplasia nor tumours, and SV40 T expression was always negative. In conclusion, transgenic mice develop large testicular tumours that are preceded by interstitial hyperplasia and microtumours. The histological and immunohistochemical phenotype of tumours (Leydig and Sertoli cell differentiation, positive 3beta-HSD, and variable AMH) suggests a mixed differentiation of somatic cells of the specialized gonadal stroma. The finding that an oncogene directed by a promoter specifically active in fetal Sertoli cells has given rise to testicular tumours of mixed differentiation is compatible with a common origin of Leydig and Sertoli cells from the specific stroma of the gonadal ridge, as supported by double labelling experiments in fetal mice showing co-localization of the transgene with Sertoli and Leydig cell markers.

Lotmaria passim (Kinetoplastea: Trypanosomatidae) in honey bees from Argentina.

Lotmaria passim (Kinetoplastea) is considered the most prevalent as well as the most virulent trypanosomatid associated to the European honey bee Apis mellifera. We used qPCR to screen for the presence of this parasite in 57 samples from ten Argentinian provinces, and were able to detect its presence throughout most of the country with 41% of the samples testing positive. In a retrospective analysis, we detected L. passim in 73% of honey bee samples from 2006 showing that this flagellate has been widely present in Argentina for at least ~15 years. Additionally, three primer sets for L. passim detection were compared, with the pair that produced smallest PCR product having the best detection capability. Finally, we also found L. passim DNA in 100% (n = 6) of samples of the mite Varroa destructor. The role of this ectoparasite in the lifecycle of Lotmaria, if any, remains unrevealed.

Improvement of Leptospira spp. diagnosis in aborted bovine fetuses by qPCR.

Leptospirosis is a disease with major economic impact on livestock industry. The objective of this work was to determine the presence of Leptospira spp. DNA by qPCR in bovine fetuses with presumptive diagnosis of leptospirosis as the cause of abortion. Leptospira spp. DNA was detected by qPCR in 11 out of 34 fetuses. These specimens (10/11) had histopathological findings in hepatic and/or renal tissues compatible with leptospirosis. qPCR detection rate (32.4 %) was higher compared with direct immuno-fluorescence antibody test (DFAT) (11.8 %). The concordance coefficient between both techniques was 0.44. qPCR is a rapid and sensitive technique for the diagnosis of leptospirosis and improved the detection rate in fetal tissues compared with DFAT. Implementation of molecular techniques may increase the accurate detection of leptospirosis as a cause of bovine abortion allowing the application of rapid therapeutic and prophylactics measures in order to reduce the impact of this zoonotic disease.

Reproductive hormones influence zinc homeostasis in the bovine cumulus-oocyte complex: Impact on intracellular zinc concentration and transporters gene expression.

Zinc (Zn) is a vital trace element for the body and its bioavailability influences numerous reproductive events. However, the mechanisms that regulate Zn homeostasis in the cumulus-oocyte complex (COC) are yet to be elucidated. The aim of this study was to investigate the role of estradiol 17-beta (E2), FSH and LH in Zn homeostasis regulation in bovine COC matured in vitro and Zn transporters gene expression. For this purpose, intracellular Zn levels in oocytes and cumulus cells (CC) were assessed using a Zn-specific fluorescent indicator. In addition, gene expression and sequencing of six Zn transporters (Slc39a6, Slc39a8, Slc39a14, Slc30a3, Slc30a7 and Slc30a9) were assessed. Our results demonstrated that the simultaneous presence of E2, FSH, and LH during oocyte maturation altered intracellular zinc levels and transporters expression in both oocytes and CC. Transporter's gene expression was different in oocytes and CC, possibly due to cell-specific changes in Zn levels during maturation. The interaction effects of Zn with hormonal treatments influenced the results. This study emphasizes that Slc39a6 is highly sensitive to hormone induction. Overall, the hormonal modulation of Zn homeostasis in the COC was evidenced. Also, a preponderant role of FSH as a modulator of Zn intracellular levels and transporter gene expression is suggested.

Characterization of multifunctional reverse micelles' interfaces using hemicyanines as molecular probes. II: Effect of the surfactant.

In this work, we have investigated the behavior of the cationic hemicyanine trans-4-[4-(dimethylamino)-styryl]-N-methylpyridinium iodide (HC) in benzene/benzyl-n-hexadecyl dimethylammonium chloride (BHDC)/water reverse micelle media using absorption and emission spectroscopy in addition to the steady-state and time-resolved fluorescence emission techniques and compare the results to those obtained in benzene/sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/water reverse micelle media (Moyano, F.; et al. J. Phys. Chem. B 2009, 113, 4284.) in order to gain more insight about reverse micelle interface properties. Our results show that HC spectroscopic behavior is completely different when dissolved in AOT or in BHDC reverse micelle media. While the dye experiences an intramolecular charge-transfer process upon excitation in the former media, in BHDC, this process is inhibited because of the cationic nature of the surfactant. Interestingly, we also show that the water properties are different for water molecules sequestrated inside of an anionic and cationic reverse micelle system. This come out because the water molecules entrapped inside of the BHDC reverse micelle media appear to be non-electron-donating because of its interaction with the cationic surfactant polar head group. On the other hand, the water molecules sequestrated inside of the AOT reverse micelle systems show its electron-donor ability enhanced in comparison with its water bulk structure. These results could also explain the lack of nucleophilicity shown by the water molecules entrapped in BHDC reverse micelle media reported in previous kinetic studies.

Characterization of multifunctional reverse micelles' interfaces using hemicyanines as molecular probes. I. Effect of the hemicyanines' structure.

In this work, we report the behavior of two different hemicyanines, trans-4-[4-(dimethylamino)styryl]-N-methylpyridinium iodide (HC) and 4-[4-(dihexadecylamino)styryl]-N-methylpyridinium iodide (DIA), in water/sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/benzene reverse micelles media using absorption and emission spectroscopy in addition to the steady-state and time-resolved fluorescence emission techniques. Our results show that the AOT reverse micelles interface has the nontrivial deaggregation property, a result that may have potential application for the preparation of dye lasers, which require a noninteracting monomeric form of the dye. Also, we show that the water interacts with a different region of the AOT moiety depending on the external organic solvent used and, in addition, we also present a nice, simple, and noteworthy method that helps to examine the presence or the absence of organized media. In conclusion, our results show that HC and DIA are powerful dyes to characterize simultaneously different interfaces' properties as they can be used to sense, at the same time, fluidity and specific interactions at the interface. These results are important because those properties are the key for molecular recognition.

First Characterization of PAH-degrading bacteria from Río de la Plata and high-resolution melting: an encouraging step toward bioremediation.

The Río de la Plata, one of the most important estuarine environments in South America that sustains valuable fisheries, is affected by PAH contamination associated with oil industry and port activities. A total of 95 bacteria with potential to degrade phenanthrene were obtained from water samples using traditional culture methods. PCR-RFLP analysis of 16S rDNA partial fragments was used as a screening tool for reducing the number of isolates during diversity studies, obtaining 42 strains with different fingerprint patterns. Phylogenetic analysis indicated that they were affiliated to 19 different genera of Gamma- and Alpha-Proteobacteria, and Actinobacteria. Some of them showed an efficient phenanthrene degradation by HPLC (between 83% and 97%) and surfactant production (between 40% and 55%). They could be an alternative for microbial selection in the degradation of PAHs in this estuarine system. In order to detect and monitor PAH-degrading bacteria in this highly productive area, rDNA amplicons of the 33 isolates, produced by PCR real time, were tested by the high-resolution melting (HRM) technique. After analyzing the generated melting curves, it was possible to accurately distinguish nine patterns corresponding to eight different genera. HRM analysis allowed a differentiation at the species level for genera Pseudomonas, Halomonas and Vibrio. The implementation of this method as a fast and sensitive scanning approach to identify PAH-degrading bacteria, avoiding the sequencing step, would mean an advance in bioremediation technologies.

Expression of Ghrelin and Its Receptor mRNA in Bovine Oocyte and Cumulus Cells.

Energy balance is regulated by ghrelin which is a neuroendocrine modulator. Ghrelin is expressed in reproductive organs. However, the role of ghrelin during in vitro maturation (IVM) and bovine preimplantational development is limited. The purpose of this study was to measure the expression of ghrelin (GHRL) and its receptor growth hormone secretagogue receptor 1A (GHS-R1A) mRNA, and determine cumulus oocyte complex (COC) viability after IVM with 0, 20, 40 and 60 pM of ghrelin. Also, pronuclear formation was recorded after in vitro fertilization (IVF). GHRL and GHS-R1A mRNA expression in oocyte and cumulus cells (CCs) was assessed using reverse transcription-polymerase chain reaction (PCR). Oocyte and CC viability were analyzed with the fluorescein diacetate fluorochrome-trypan blue technique. Pronuclear formation was determined 18 hours after IVF with Hoechst 33342. The results demonstrated that ghrelin mRNA is present in oocyte and CCs before and after 24 hours IVM with all treatments. Ghrelin receptor, GHS-R1A, was only detected in oocytes and CCs after 24 hours IVM with 20, 40 and 60 pM of ghrelin. Oocyte viability was not significantly different (P=0.77) among treatments. However, CC viability was significantly lower (P=0.04) when COCs were matured with ghrelin (77.65, 72.10, 66.32 and 46.86% for 0, 20, 40, and 60 pM of ghrelin, respectively). The chance of two pronuclei forming were higher (P=0.03) when ghrelin was not be added to the IVM medium. We found that ghrelin negatively impacts CC viability and pronuclear formation.

Towards Precision Nutrition: A Novel Concept Linking Phytochemicals, Immune Response and Honey Bee Health.

The high annual losses of managed honey bees (Apis mellifera) has attracted intensive attention, and scientists have dedicated much effort trying to identify the stresses affecting bees. There are, however, no simple answers; rather, research suggests multifactorial effects. Several works have been reported highlighting the relationship between bees' immunosuppression and the effects of malnutrition, parasites, pathogens, agrochemical and beekeeping pesticides exposure, forage dearth and cold stress. Here we analyze a possible connection between immunity-related signaling pathways that could be involved in the response to the stress resulted from Varroa-virus association and cold stress during winter. The analysis was made understanding the honey bee as a superorganism, where individuals are integrated and interacting within the colony, going from social to individual immune responses. We propose the term "Precision Nutrition" as a way to think and study bees' nutrition in the search for key molecules which would be able to strengthen colonies' responses to any or all of those stresses combined.

Laurus nobilis L. Extracts against Paenibacillus larvae: Antimicrobial activity, antioxidant capacity, hygienic behavior and colony strength.

The aim of this work was to compare the antimicrobial activity against Paenibacillus larvae and the antioxidant capacity of two Laurus nobilis L. extracts obtained by different extraction methods. The hydroalcoholic extract was moreover added as supplementary diet to bees in field conditions to test behavioural effects and colony strength. Both laurel extracts were subjected to different phytochemical analysis to identify their bioactive compounds. Antimicrobial activity was analyzed by the minimal inhibitory concentration (MIC) determination by means the agar dilution method. The hydroalcoholic extract (HE) was able to inhibit the bacterial growth of all P. larvae strains, with 580 µg/mL mean value. This better antibacterial activity in relation to the essential oil (EO) could be explained by the presence of some phenolic compounds, such as flavonoids, evidenced by characteristic bands resulting from the Fourier Transform Infrared Spectroscopy (FTIR) analysis. Antioxidant activities of the extracts were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging ability and ferric reducing antioxidant power (FRAP) assays. The HE showed the highest antioxidant activity as measured by DPPH, with IC50 values of 257 ±â€¯12 µg/mL. The FRAP assay method showed that the HE was 3-fold more effective reducing agent than the EO. When the bee colonies were supplied with laurel HE in sugar paste an improvement in their general condition was noticed, although neither the hygienic behavior nor the proportions of the breeding cells varied statistically due to the treatment. In conclusion, the inhibition power against P. larvae attributable to the phenolic compounds, the antioxidant capacity of the HE, and the non-lethal effects on adult honey bees on field trials suggest the HE of laurel as a promising substance for control American foulbrood disease.