Emerging Hyalomma lusitanicum: From identification to vectorial role and integrated control.

In the Mediterranean basin, the tick species Hyalomma lusitanicum Koch stands out among other species of the Hyalomma genus due to its wide distribution, and there is great concern about its potential role as a vector and/or reservoir and its continuous expansion to new areas because of climate warming and human and other animal movements. This review aims to consolidate all the information on H. lusitanicum, including taxonomy and evolution, morphological and molecular identification, life cycle, sampling methods, rearing under laboratory conditions, ecology, hosts, geographical distribution, seasonality, vector role and control methods. The availability of adequate data is extremely relevant to the development of appropriate control strategies in areas where this tick is currently distributed as well as in new areas where it could become established in the near future.

Study of pyrethroid resistance mutations in populations of Varroa destructor across Spain.

The Varroa destructor mite is a serious worldwide pest of honeybees that is usually controlled with pyrethroid-based acaricides. However, the intensive use of these substances over the past decades has led to the development of resistance in these mites. Here, Varroa samples collected between 2006 and 2021 from apiaries across Spain were studied to evaluate the presence of mutations producing pyrethroid resistance, particularly those in the gene encoding the voltage-gated sodium channel (VGSC). Genotyping of the IIS4-IIS5 region of this gene detected the L925V (Leucine 'CTG' to valine 'GTG') mutation at position 925 and confirmed the presence of the M918L (Methionine 'ATG' to Leucine 'TTG') mutation at position 918 in these Spanish Varroa mites. Interestingly, the M918L mutation was always found in combination with L925V, both of which were always homozygous. Over and above the high frequency of pyrethroid-resistant mutations in Spanish Varroa populations, this apparently recent association of the M918L and L925V point mutations is a combination that appears to trigger greater resistance than that produced by L925V alone.

Gene Expression and Fatty Acid Profiling in Longissimus thoracis Muscle, Subcutaneous Fat, and Liver of Light Lambs in Response to Concentrate or Alfalfa Grazing.

A better understanding of gene expression and metabolic pathways in response to a feeding system is critical for identifying key physiological processes and genes associated with polyunsaturated fatty acid (PUFA) content in lamb meat. The main objective of this study was to investigate transcriptional changes in L. thoracis (LT) muscle, liver, and subcutaneous fat (SF) of lambs that grazed alfalfa (ALF) and concentrate-fed (CON) slaughtered at 23 kg and using the Affymetrix Ovine Gene 1.1 ST whole-genome array. The study also evaluated the relationship between meat traits in LT muscle, including color, pigments and lipid oxidation during 7 days of display, α-tocopherol content, intramuscular fat (IMF) content and the fatty acid (FA) profile. Lambs that grazed on alfalfa had a greater α-tocopherol concentration in plasma than CON lambs (P < 0.05). The treatment did not affect the IMF content, meat color or pigments (P > 0.05). Grazing increased the α-tocopherol content (P < 0.001) and decreased lipid oxidation on day 7 of display (P < 0.05) in LT muscle. The ALF group contained a greater amount of conjugated linoleic acid (CLA), C18:3 n-3, C20:5 n-3, C22:5 n-3, and C22:6 n-3 than did the CON group (P < 0.05). We identified 41, 96 and four genes differentially expressed in LT muscle, liver, and subcutaneous fat, respectively. The most enriched biological processes in LT muscle were skeletal muscle tissue development, being the genes related to catabolic and lipid processes downregulated, except for CPT1B, which was upregulated in the ALF lambs. Animals grazing alfalfa had lower expression of desaturase enzymes in the liver (FADS1 and FADS2), which regulate unsaturation of fatty acids and are directly involved in the metabolism of n-3 PUFA series. The results found in the current study showed that ingesting diets richer in n-3 PUFA might have negative effects on the de novo synthesis of n-3 PUFA by downregulating the FADS1 and FADS2 expression. However, feeding diets poorer in n-3 PUFA can promote fatty acid desaturation, which makes these two genes attractive candidates for altering the content of PUFAs in meat.

Antifungal activity of the essential oil obtained from Cryptocarya alba against infection in honey bees by Nosema ceranae.

The honeybee disease nosemosis type C is a serious problem since its causative agent, microsporidium Nosema ceranae, is widespread among adult honey bees. Some of the feasible alternative treatments that are used to control this disease are plant extracts. The aim of the present work was to evaluate the effects of essential oils of Chilean plant species, such as Cryptocarya alba, which is used against N. ceranae, and to identify and quantify the majority active compounds in the EO as well as their potential use for the control of nosemosis. Essential oils were obtained using the stripping steam technique with Clevenger equipment and were subsequently analyzed by Gas chromatography-mass spectrometry. Mortality was recorded daily over at least 8days as worker honeybees were exposed to a range of doses of EO dispersed in a sucrose solution. C. alba oil appears to be nontoxic to A. mellifera adults at the tested concentration (the same concentration inhibits the growth of N. ceranae), showing that this oil can be used for the treatment of nosemosis. EO effectiveness was demonstrated against N. ceranae by calculating the percentage of decrease in infected bees from untreated infected groups vs infected groups treated with EO or the reference drug fumagillin. It was determined that a dose of 4µg EO/bee was most effective in controlling N. ceranae development. We determined innocuous doses of C. alba essential oil for honeybees. We demonstrated the antifungal activity of C. alba EO at 4µg/bee against N. ceranae and compared it to its major monoterpenes, such as ß-phellandrene (20µg/bee), eucalyptol (20µg/bee) and α-terpineol (20µg/bee). The major compounds of C. alba EO, α-terpineol, eucalyptol and ß-phellandrene, had significant effects against Apis mellifera infection by N. ceranae, but the antifungal effect of the complete essential oil on N. ceranae was larger than the effect of α-terpineol, eucalyptol or ß- phellandrene separately, showing that C. alba oil may be a candidate for the treatment or prevention of nosemosis.

Characterization of Nosema ceranae Genetic Variants from Different Geographic Origins.

In recent years, large-scale colony losses of honey bees (Apis mellifera) have been reported and the infection with the microsporidia Nosema ceranae has been involved. However, the effect of N. ceranae at the colony level and its role in colony losses vary in different geographic areas. This difference may be related to the presence of multiple N. ceranae genetic variants resulting in different biological consequences. In this study, we analyzed the genetic diversity of 75 N. ceranae samples obtained from 13 countries and Hawaii through inter-sequence single repetition (ISSR) and evaluated if two of these genetic variants triggered different immune responses when infecting Apis mellifera iberiensis. The genetic diversity analysis showed that 41% of the samples had the same DNA amplification pattern, including samples from most European countries except Spain, while the remaining samples showed high variability. Infection assays were performed to analyze the infection levels and the immune response of bees infected with N. ceranae from Spain and Uruguay. The infected bees presented similar infection levels, and both isolates downregulated the expression of abaecin, confirming the ability of the microsporidia to depress the immune response. Only N. ceranae from Uruguay downregulated the expression level of imd compared to control bees. On the other hand, both genetic variants triggered different expression levels of lysozyme. As imd and lysozyme play important roles in the response to pathogens, these results could reflect differences in the biological consequences of N. ceranae variants in A. mellifera infection.

Circulating microRNAs in Huntington's disease: Emerging mediators in metabolic impairment.

Huntington's disease (HD) is a hereditary neurodegenerative disease, with peripheral consequences that negatively contribute to quality of life. Circulating microRNAs (cmiRNAs) are being explored for their roles in intercellular communication and gene expression regulation, which allows gaining insight into the regulation of crosstalk between neuronal and peripheral tissues. Here, we explore the cmiRNA profile of plasma samples from fifteen symptomatic patients, with 40-45 CAG repeats in the HTT gene, and seven healthy matched controls. Isolated miRNAs from plasma samples were run against human miRNome panels, which have sequences for 752 human mature miRNAs. We found that 168 cmiRNAs are altered in symptomatic patients. Considering Bonferroni's correction, miR-877-5p, miR-223-3p, miR-223-5p, miR-30d-5p, miR-128, miR-22-5p, miR-222-3p, miR-338-3p, miR-130b-3p, miR-425-5p, miR-628-3p, miR-361-5p, miR-942 are significantly increased in HD patients as compared with controls. Moreover, after patient's organization according to approved HD scales, miR-122-5p is significantly decreased in HD patients with Unified Huntington's Disease Rating Scale >24, whereas an increase in miR-100-5p levels and a decrease in miR-641 and miR-330-3p levels were recorded when patients were rearranged by Total Functional Capacity. These results suggest that cmiRNA profile could be further modified by disease progression, making cmiRNAs useful as monitoring biomarkers. Analysis of target genes indicated a general overexpression of cmiRNAs implicated in metabolism regulation. Profiling cmiRNA of HD subjects opens the possibility of personalized therapies for different groups of HD patients, based on disease modifiers: regulation of altered pathways might contribute to not only alleviate disease symptoms, but also influence HD progression.

High prevalence and infection levels of Nosema ceranae in bumblebees Bombus atratus and Bombus bellicosus from Uruguay.

Nosema ceranae is one of the most prevalent pathogens in Apis mellifera and has recently been found in multiple host species including several species of bumblebees. Prevalence and infection intensity of N. ceranae was determined in two species of native bumblebees from Uruguay. Nosema ceranae was the only microsporidia identified and mean prevalence was 72% in Bombus atratus and 63% in Bombus bellicosus, values much higher than those reported elsewhere. The presence of this pathogen in bumblebees may be threatening not only for bumblebee populations, but also to the rest of the native pollinator community and to honeybees.

Recent worldwide expansion of Nosema ceranae (Microsporidia) in Apis mellifera populations inferred from multilocus patterns of genetic variation.

Nosema ceranae has been found infecting Apismellifera colonies with increasing frequency and it now represents a major threat to the health and long-term survival of these honeybees worldwide. However, so far little is known about the population genetics of this parasite. Here, we describe the patterns of genetic variation at three genomic loci in a collection of isolates from all over the world. Our main findings are: (i) the levels of genetic polymorphism (πS≈1%) do not vary significantly across its distribution range, (ii) there is substantial evidence for recombination among haplotypes, (iii) the best part of the observed genetic variance corresponds to differences within bee colonies (up to 88% of the total variance), (iv) parasites collected from Asian honeybees (Apis cerana and Apis florea) display significant differentiation from those obtained from Apismellifera (8-16% of the total variance, p<0.01) and (v) there is a significant excess of low frequency variants over neutral expectations among samples obtained from A. mellifera, but not from Asian honeybees. Overall these results are consistent with a recent colonization and rapid expansion of N. ceranae throughout A. mellifera colonies.

Is Acarapis woodi a single species? A new PCR protocol to evaluate its prevalence.

Acarapisosis is a disease of the adult honey bee Apis mellifera L., caused by the tracheal mite Acarapis woodi (Rennie), that affects the prothoracic tracheas of worker honey bees. Although it is not usually considered a real problem for honey bee colonies in southern Europe (mainly Spain and Greece), where the majority of professional beekeepers are located in Europe, recent works have reported the constant presence of this mite in this area, making it a potential cofactor for colony losses. In this study, we developed a specific PCR diagnostic tool that improves the techniques used so far and allowed us to confirm the presence of this parasite in Spain, urging the need to monitor its prevalence and implications in the health of the colonies. Indeed, in a total of 635 apiaries analysed, the prevalence of A. woodi in 2010 was 8.3 and 4 % in 2011. The mite is present in bee colonies over time and should not be underestimated as a possible cofactor in the collapse of bee colonies. Additionally, some positive samples were cloned so a genetic analysis on the diversity within A. woodi isolates was also approached. This allowed us to identify different genetic variants within an isolate, even when they were present at low frequencies. And this genetic analysis revealed the existence of a different clade of Acarapis sequences that could represent a new species or subspecies, although more research is required to verify the identity of this novel lineage at genetic and morphological level.

Flow cytometry analysis of Nosema species to assess spore viability and longevity.

Nosema apis and Nosema ceranae are microsporidia which present resistant spores for the transmission stage (environmental spores) that play an important role for epidemiology and for laboratory studies of honey bee microsporidiosis. In this study, the long-term longevity of N. apis and N. ceranae spores exposed to 4 °C, room temperature (mean 25 °C) and 35 °C for 6-month long and to -20 °C for 10-month long has been assessed by flow cytometry. Storage temperature and the length of storage duration had adverse effects on spore viability of both Nosema spores, with significant differences between the two species. The greatest increase in spore mortality was observed in N. apis spores stored at 33 °C (64, 89%) and in N. ceranae spores at -20 °C (53.55%) and at 33 °C (51.97%). For N. ceranae spores at -20 °C, the loss in viability was very quick, getting an increase over 20% just after 6 days of exposure. Results on viability were confirmed by the infectivity tests where the lowest infectivity for N. ceranae was observed with spores stored for 10 months at -20 °C (79%; P < 0.05) and for N. apis with spores stored at 33 °C (71%; P < 0.05). For both Nosema species, the best storage temperatures were 25 and 4 °C, especially for N. apis that was almost unaffected at those temperatures.

Liquid chromatography coupled to ion trap-tandem mass spectrometry to evaluate juvenile hormone III levels in bee hemolymph from Nosema spp. infected colonies.

It has been described a fast, simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure juvenile hormone III (JH III), which was used to study of the effects of Nosema spp. infection on JH III levels in bee hemolymph. Honey bee hemolymph was extracted by centrifugation and mixed with a solution of phenylthiourea in methanol. This mixture was then centrifuged and the supernatant removed and evaporated to dryness. The residue was reconstituted in methanol containing the internal standard (methoprene) and injected onto an LC-MS/MS (ion-trap) system coupled to electrospray ionization (ESI) in positive mode. Chromatography was performed on a Synergi Hydro-RP column (4 µm, 30 mm × 4.60 mm i.d.) using a mobile phase of 20 mM ammonium formate and methanol in binary gradient elution mode. The method was fully validated and it was found to be selective, linear from 15 to 14,562 pg/µL, precise and accurate, with %RSD values below 5%. The limits of detection and quantification were: LOD, 6 pg/µL; LOQ, 15 pg/µL. Finally, the proposed LC-MS/MS method was used to analyze JH III levels in the hemolymph of worker honey bees (Apis mellifera iberiensis) experimentally infected with different Nosema spp. (Nosema apis, Spanish and Dutch Nosema ceranae strains). The highest concentrations of JH III were detected in hemolymph from bees infected with Spanish N. ceranae.

Low prevalence of honeybee viruses in Spain during 2006 and 2007.

RNA viruses that affect honeybees have been involved in colony losses reported around the world. The aim of the present work was to evaluate the prevalence and distribution of honeybee viruses during 2006-2007 in Spanish professional apiaries, and their association with colony losses. Four hundred and fifty-six samples from apiaries located in different geographic regions of Spain were analyzed. Thirty-seven percent of the samples had viral presence. Most (80%) had one virus and 20% two different viruses. All the analyzed viruses, Deformed Wing Virus (DWV), Israeli Acute Paralysis Virus (IAPV), Black Queen Cell Virus (BQCV), Sacbrood Virus (SBV) and Kashmir Bee Virus (KBV) were detected, but detection rates were lower than expected. According to these results and considering the high prevalence of other honeybee pathogens in Spain, the role of viruses in colony losses in Spain may be discussed.

Determination of tylosins A, B, C and D in bee larvae by liquid chromatography coupled to ion trap-tandem mass spectrometry.

A LC-MS/MS method has been developed to simultaneously quantify tylosins A, B, C and D in bee larvae, compounds currently used to treat one of the most lethal diseases affecting honey bees around the world, American Foulbrood (AFB). The influence of different aqueous media, temperature and light exposure on the stability of these four compounds was studied. The analytes were extracted from bee larvae with methanol and chromatographic separation was achieved on a Luna C(18) (150 × 4.6 mm i.d.) using a ternary gradient composed of a diluted formic acid, methanol and acetonitrile mobile phase. To facilitate sampling, bee larvae were initially dried at 60°C for 4h and afterwards, they were diluted to avoid problems of pressure. MSD-Ion Trap detection was employed with electrospray ionization (ESI). The calibration curves were linear over a wide range of concentrations and the method was validated as sensitive, precise and accurate within the limits of quantification (LOQ, 1.4-4.0 ng/g). The validated method was successfully employed to study bee larvae in field tests of bee hives treated with two formulations containing tylosin. In both cases it was evident that the minimal inhibitory concentration (MIC) had been reached.

Evaluation of large-scale dissemination of Nosema ceranae spores by European bee-eaters Merops apiaster.

Identification of transmission routes and of factors affecting the spatial positions of pathogens, hosts and vectors is basic to an adequate disease management. Nosema ceranae is a Microsporidian recently described as a parasite of Apis mellifera honeybees and is currently considered the aetiological agent of an emergent illness named nosemosis type C. In this article we evaluate the role of a bird species, the European bee-eater, Merops apiaster, as a large-scale dispersive agent of N. ceranae. We found a high prevalence of viable spores of N. ceranae in pellets regurgitated by bee-eaters in different locations in the Iberian Peninsula, Central Europe and central Asia. In contrast, spores of Nosema apis, considered till recently the most common microsporidium infecting honeybees, were detected in a single locality and Nosema bombi spores were not noticed. Since non-viable spores were also found in bee-eater nests from different locations, this bird species could also reduce the fraction of infected insects by withdrawing pathogens from the colonies. We conclude that bee-eater mobility and migration may have played an important role in the transmission of the pathogen N. ceranae.

Overview of pesticide residues in stored pollen and their potential effect on bee colony (Apis mellifera) losses in Spain.

In the last decade, an increase in honey bee (Apis mellifera L.) colony losses has been reported in several countries. The causes of this decline are still not clear. This study was set out to evaluate the pesticide residues in stored pollen from honey bee colonies and their possible impact on honey bee losses in Spain. In total, 1,021 professional apiaries were randomly selected. All pollen samples were subjected to multiresidue analysis by gas chromatography-mass spectrometry (MS) and liquid chromatography-MS; moreover, specific methods were applied for neonicotinoids and fipronil. A palynological analysis also was carried out to confirm the type of foraging crop. Pesticide residues were detected in 42% of samples collected in spring, and only in 31% of samples collected in autumn. Fluvalinate and chlorfenvinphos were the most frequently detected pesticides in the analyzed samples. Fipronil was detected in 3.7% of all the spring samples but never in autumn samples, and neonicotinoid residues were not detected. More than 47.8% of stored pollen samples belonged to wild vegetation, and sunflower (Heliantus spp.) pollen was only detected in 10.4% of the samples. A direct relation between pesticide residues found in stored pollen samples and colony losses was not evident accordingly to the obtained results. Further studies are necessary to determine the possible role of the most frequent and abundant pesticides (such as acaricides) and the synergism among them and with other pathogens more prevalent in Spain.

High-level resistance of Nosema ceranae, a parasite of the honeybee, to temperature and desiccation.

Resistance of Nosema ceranae to different exposure conditions has been evaluated by using Sytox green and DAPI (4',6-diamidino-2-phenylindole) to test spore viability. High thermotolerance at 60 and 35 degrees C and resistance to desiccation were observed. However, a significant decrease in viability after freezing and a rapid degeneration of spores maintained at 4 degrees C were also detected.

Analysis of stage-specific and shared antigens derived from Rhipicephalus sanguineus by electrophoresis and western blotting.

We carried out an SDS-PAGE analysis of antigens of Rhipicephalus sanguineus using extracts of eggs (EE), larvae (LE), nymphs (NE), male salivary glands (MSGE), male midguts (MME), female salivary glands (FSGE) and female midguts (FME). Under non-reducing conditions a common band of about 205 kDa was observed. EE, LE and NE extracts showed groups of bands between 150 and 75 kDa. A protein pattern was observed in FSGE extract with a group of bands between 75 and 50 kDa and four bands between 15 and 6.5 kDa. In this case an apparently exclusive band of molecular weight about 25 kDa was observed. Under reducing conditions similarities between LE and NE extracts increased, separating from the EE pattern. On the other hand, we have determined the presence of stage-specific and common antigens on EE, LE, NE, MSGE, MME, FSGE and FME extracts of R.sanguineus by means of immunoblots using polyclonal sera of rabbits infested with larvae, nymphs or adults of this tick. EE extract was only recognized by the anti-larva sera. Higher reactivity was observed when the extracts were tested with anti-adult sera. In these experiments a very prominent band of molecular weight about 45 kDa was detected. This band was not observed under reducing conditions. Higher reactivity with anti-adult sera was observed against FSGE extract.

Immunological response in rabbits infested with Rhipicephalus sanguineus.

In order to clarify the importance of humoral antibody in host resistance to ticks, in the present work we studied the immunological response of rabbits infested with larvae, nymphs or adults of Rhipicephalus sanguineus, using extracts of eggs (EE), larvae (LE), nymphs (NE), male salivary glands (MSGE), male midguts (MME), female salivary glands (FSGE) and female midguts (FME). When serum from rabbits infested with larvae or nymphs was tested using enzyme-linked immunosorbent assay, no reactions were observed with any of the extracts including the homologous LE or NE. In sera from rabbits infested with adult ticks, the reactions were observed in both homologous (MSGE, MME, FSGE and FME) and heterologous (EE, LE and NE) system. However, differences were seen regarding the type of antigen used. When the experiment was carried out using extracts from adults higher responses were found. With FSGE and FME antigens, antibody levels were systematically higher than those observed when MSGE and MME were used.