Propolis components accountable for bactericidal accomplishment and antibiofilm activity

This study aimed at evaluating antimicrobial and antibiofilm activity of phenolic compounds present in propolis ethanol extracts (PEE). Seventy per cent ethanol extracts from seven types of propolis, one Green, two Red and four Brown collected in four Brazilian States were prepared and total phenolics, flavonoids, tannins and anthocyanins were assessed by high-performance liquid chromatography (HPLC). Minimum bactericidal concentration (MBC) and inhibitor effect on Staphylococcus aureus biofilm formation and capacity to disrupt established biofilms were assessed towards eight S. aureus isolates from milk of small ruminants with mastitis, one methicillin-resistant S. aureus (MRSA) and S. aureus ATCC 25923. To evaluate different propolis components accountability for bactericidal accomplishment and antibiofilm activity, the results were analysed by the non-parametric Spearman coefficient. Results of phenolic compounds were 216,21 to 312,08 gallic acid milligram equivalent per extract gram (mg EGA/g) of total phenolics, 55,08 to 140,6 quercetin milligram equivalent per extract gram (mg EQ/g) of flavonoids, 118,51 to 3766,16 catechin milligram equivalent per extract gram (mg EC/g) of tannins and 1,03 to 8,39 milligram per extract gram (mg/g) of anthocyanins. Red1 and Red2 showed higher tannin contents, while Red2 exhibited superior amount of anthocyanins and total phenolics. Brown3 presented higher flavonoid quantity. Green, Red1 and Red2 PEE showed the lowest levels of flavonoids, but the higher antimicrobial activity. Most PEE exhibit bactericidal activity at a concentration of 1.6 mg/mL. Brown4 PEE showed the worst capacity to inhibit S. aureus. Green PEE showed to be the most efficient in both preventing and disrupting biofilm. All PEE studied exhibited a better inhibitory activity prior-to than post-biofilm formation. According to non-parametric Spearman correlation analysis, there seems to be a significant negative correlation between the ability to disrupt biofilm and both tannins and anthocyanins contents.

A holistic approach for the investigation of bee health

Despite current evidence is in strong disagreement with an emergency for the conservation of Apis mellifera, great concern is related to profitability of beekeeping operations. A growing involvement of veterinary science in addressing bee health topics will therefore be fundamental to preserve and protect the entire sector. The experiments in this thesis focused on two different and interdependent levels related to bee health: the biochemical level and the parasitological level. At the biochemical level the impact of plant protection products on bee physiology and survival was studied, elucidating synergistic interactions between poor nutrition and pesticide exposure in A. mellifera and between an insecticide and a fungicide in Osmia bicornis. Moreover, an innovative fingerprinting approach on honey bee haemolymph was applied to detect population imbalances in the hive. The control of Varroa infestations was studied both at the biochemical and parasitological level. A panel of biomarkers in honey bee haemolymph was applied to compare different mite control protocols. This resulted in relevant indications for beekeeping operations pursuing the least impact on nutritional status of the colonies. To guide the decision making of beekeepers, a new formic acid evaporator was tested in comparison with a more established one. Considering its widespread distribution in the country, efforts were directed also towards N. ceranae. In particular, the pivotal aspect of diagnosis was studied, proposing a new qPCR method to overcome some limits of the existing ones. In conclusion, this works fills some of the knowledge gaps of the beekeeping sector. However, many of them still need to be addressed and the upcoming menaces of climate change and dispersal of pathogens via globalization should be targeted by research efforts in the near future. Therefore, a multifaceted vision of bee health is of capital importance, aware of the complementarity of reductionist and holistic approaches.

Applied genomics to monitor the genetic diversity and integrity of honey bee (Apis mellifera) populations and define sustainable strategies to benefit the beekeeping sector

Honey bees are considered keystone species in ecosystem, the effect of harmful pesticides for the honey bees, the action of extreme climatic waves and their consequence on honey bees health can cause the loss of many colonies which could contribute to the reduction of the effective population size and incentive the use of non-autochthonous queens to replace dead colonies. Over the last decades, the use of non-ligustica bee subspecies in Italy has increased and together with the mentioned phenomena exposed native honey bees to hybridization, laeding to a dramatic loss of genetic erosion and admixture. Healthy genetic diversity within honey bee populations is critical to provide tolerance and resistance to current and future threatening. Nowadays it is urgent to design strategies for the conservation of local subspecies and their valorisation on a productive scale. In this Thesis we applied genomics tool for the analysis of the genetic diversity and the genomic integrity of honey bee populations in Italy are described. In this work mtDNA based methods are presented using honey bee DNA or honey eDNA as source of information of the genetic diversity of A. mellifera at different level. Taken together, the results derived from these studies should enlarge the knowledge of the genetic diversity and integrity of the honey bee populations in Italy, filling the gap of information necessary to design efficient conservation programmes. Furthermore, the methods presented in these works will provide a tool for the honey authentication to sustain and valorise beekeeping products and sector against frauds.

Assessing the relationship between bee/flower diversity and vegetation structural heterogeneity through UAV photogrammetry

The ecosystem services provided by bees are very important. Factors as habitat fragmentation, intensive agriculture and climate change are contributing to the decline of bee populations. The use of remote sensing could be a useful tool for the recognition of sites with a high diversity, before performing a more expensive survey in the field. In this study the ability of Unmanned Aerial Vehicles (UAV) images to estimate biodiversity at local scale has been analysed testing the concept of the Height Variation Hypothesis (HVH). This approach states that, the higher the vegetation height heterogeneity (HH) measured by remote sensing information, the higher the vertical complexity and the higher vegetation species diversity. In this thesis the concept has been brought to a higher level, in order to understand if the vegetation HH can be considered a proxy also of bee species diversity and abundance. We tested this approach collecting field data on bees/flowers and RGB images through an UAV campaign in 30 grasslands in the South of the Netherlands. The Canopy Height Model (CHM) were derived through the photogrammetry technique "Structure from Motion" (SfM) with resolutions of 10cm, 25cm, 50cm. Successively, the HH assessed on the CHM using the Rao's Q heterogeneity index was correlated to the field data (bee abundance, diversity and bee/flower species richness). The correlations were all positive and significant. The highest R2 values were found when the HH was calculated at 10cm and correlated to bee species richness (R2 = 0.41) and Shannon’s H index (R2 = 0.38). Using a lower spatial resolution the goodness of fit slightly decreases. For flower species richness the R2 ranged between 0.36 to 0.39. Our results suggest that methods based on the concept behind the HVH, in this case deriving information of HH from UAV data, can be developed into valuable tools for large-scale, standardized and cost-effective monitoring of flower diversity and of the habitat quality for bees.

Flower cover and bee diversity estimate by drone RGB images

Worldwide, biodiversity is decreasing due to climate change, habitat fragmentation and agricultural intensification. Bees are essential crops pollinator, but their abundance and diversity are decreasing as well. For their conservation, it is necessary to assess the status of bee population. Field data collection methods are expensive and time consuming thus, recently, new methods based on remote sensing are used. In this study we tested the possibility of using flower cover diversity estimated by UAV images (FCD-UAV) to assess bee diversity and abundance in 10 agricultural meadows in the Netherlands. In order to do so, field data of flower and bee diversity and abundance were collected during a campaign in May 2021. Furthermore, RGB images of the areas have been collected using Unmanned Aerial Vehicle (UAV) and post-processed into orthomosaics. Lastly, Random Forest machine learning algorithm was applied to estimate FCD of the species detected in each field. Resulting FCD was expressed with Shannon and Simpson diversity indices, which were successively correlated to bee Shannon and Simpson diversity indices, abundance and species richness. The results showed a positive relationship between FCD-UAV and in-situ collected data about bee diversity, evaluated with Shannon index, abundance and species richness. The strongest relationship was found between FCD (Shannon Index) and bee abundance with R2=0.52. Following, good correlations were found with bee species richness (R2=0.39) and bee diversity (R2=0.37). R2 values of the relationship between FCD (Simpson Index) and bee abundance, species richness and diversity were slightly inferior (0.45, 0.37 and 0.35, respectively). Our results suggest that the proposed method based on the coupling of UAV imagery and machine learning for the assessment of flower species diversity could be developed into valuable tools for large-scale, standardized and cost-effective monitoring of flower cover and of the habitat quality for bees.

Própolis do sudeste e nordeste do Brasil: influência da sazonalidade na atividade antibacteriana e composição fenólica

The composition and biological activities of propolis, a resinous hive product collected by honeybees from various plant sources, depends on various factors such as season and vegetation of the area. The aim of this study was to evaluate the influence of the seasonal effect on the ethanolic extracts of Brazilian propolis (EEP) type 6 and type 12, collected during 6 months in terms of antibacterial activity and phenolic composition. The antimicrobial properties were evaluated by MIC and MBC on S. mutans Ingbritt 1600 and the profile of chemical composition by UV-visible spectrophotometry, HPLC-RF and GC-MS. The results demonstrated that the season in which propolis is collected influences its chemical composition, resulting in modifications in its antibacterial activity.

Reproductive biology of Davilla kunthii A. St-Hil. (Dilleniaceae) in Central Amazonia

This survey aimed at describing the interactions of floral visitors and Davilla kunthii A. St.-Hil. as well as characteristics of its reproductive biology in Itacoatiara, state of Amazonas, Brazil. Tests of the breeding system were performed. The guild of visitors was described according to richness, abundance, relative frequency and constancy. The breeding system tests indicated that D. kunthii is self-compatible. The pollination system was characterized as generalist, with 39 visitor species, from three different orders. Bees were the main group of pollinators, thus some behavioural aspects were described. Th e period of highest foraging activity was between 7 and 10 am. Some species presented agonistic and monopolistic behaviour. Given the behaviour and destructive potential, the Curculionidae seem to have a greater impact as seed predators than pollinators.

ESTUDO DA PREPARAÇÃO DOS EXTRATOS DE PRÓPOLIS E SUAS APLICAÇÕES

Ethanolic extracts from propolis were performed by using lhe water and vaflous coneentrations of etanol as solvent. The extracts were investigated by measurement of absorption spectruin with Uv-spectrophotometer (UV-scanning), reversed phase-high performance thin-layer chromatography, Reversed phase-HPLC. Maximum absorption of ali extracts was 290 nm, resembling flavonoid compounds and 80% ethanolic extract showed highest absorption at 290 nm. The most isosakuranetin, quercefin, and kaempferol were extracted from mixtures of propolis and 60% etanol, whereas 70% etanol extracted te most pinocembrin and sakuranetin, but 80% etanol extracted more kaempferide, acacetin, and isorhamnetin from propolis. The 60 to 80% ethanolic extracts ofpropolis inhibited highly to microbial growth and 70 and 80% ethanolic extracts showed lhe greatest antioxidant activity and 80% ethanolic extract inhibited highly to hyaluronidase activity.

The Sperm Of Hexagenia (pseudeatonica) Albivitta Walker (ephemeroptera: Fossoriae: Ephemeridae)

This study describes the sperm morphology of the mayfly Hexagenia (Pseudeatonica) albivitta (Ephemeroptera). Its spermatozoon measures approximately 30 μm of which 9 μm corresponds to the head. The head is composed of an approximately round acrosomal vesicle and a cylindrical nucleus. The nucleus has two concavities, one in the anterior tip, where the acrosomal vesicle is inserted and a deeper one at its base, where the flagellum components are inserted. The flagellum is composed of an axoneme, a mitochondrion and a dense rod adjacent to the mitochondrion. A centriolar adjunct is also observed surrounding the axoneme in the initial portion of the flagellum and extends along the flagellum for at least 2 μm, surrounding the axoneme in a half-moon shape. The axoneme is the longest component of the flagellum, and it follows the 9+9+0 pattern, with no central pair of microtubules. At the posterior region of the flagellum, the mitochondrion has a dumb-bell shape in cross sections that, together with the rectangular mitochondrial-associated rod, is responsible for the flattened shape of the flagellum. An internal membrane is observed surrounding both mitochondrion and its associated structure.

Determination of the maximum inhibitory dilution of cetylpyridinium chloride-based mouthwashes against staphylococcus aureus: an in vitro study

The aim of this in vitro study was to determine the maximum inhibitory dilution (MID) of four cetylpyridinium chloride (CPC)-based mouthwashes: CPC+Propolis, CPC+Malva, CPC+Eucaliptol+Juá+Romã+Propolis (Natural Honey®) and CPC (Cepacol®), against 28 Staphylococcus aureus field strains, using the agar dilution method. Decimal dilutions ranging from 1/10 to 1/655,360 were prepared and added to Mueller Hinton Agar. Strains were inoculated using Steers multipoint inoculator. The inocula were seeded onto the surface of the culture medium in Petri dishes containing different dilutions of the mouthwashes. The dishes were incubated at 37ºC for 24 h. For readings, the MID was considered as the maximum dilution of mouthwash still capable of inhibiting microbial growth. The obtained data showed that CPC+Propolis had antimicrobial activity against 27 strains at 1/320 dilution and against all 28 strains at 1/160 dilution, CPC+Malva inhibited the growth of all 28 strains at 1/320 dilution, CPC+Eucaliptol+Juá+Romã+Propolis inhibited the growth of 2 strains at 1/640 dilution and all 28 strains at 1/320 dilution, and Cepacol® showed antimicrobial activity against 3 strains at 1/320 dilution and against all 28 strains at 1/160 dilution. Data were submitted to Kruskal-Wallis test, showing that the MID of Cepacol® was lower than that determined for the other products (p<0.05). In conclusion, CPC-mouthwashes showed antimicrobial activity against S. aureus and the addition of other substances to CPC improved its antimicrobial effect.

As abelhas, os serviços ecossistêmicos e o Código Florestal Brasileiro

As abelhas são consideradas os principais polinizadores em ambientes naturais e agrícolas. Esse serviço ecossistêmico é essencial para a manutenção das populações selvagens de plantas e para a produção de alimento nos ambientes agrícolas e está ameaçado em várias regiões do mundo. O desmatamento é uma das causas principais porque ele afeta as populações de abelhas. A conservação das florestas é necessária para a manutenção das populações de abelhas e da polinização nas paisagens agrícolas.

Diversidade de abelhas (Hymenoptera, Apidae) ao longo de um gradiente latitudinal na Mata Atlântica

A Mata Atlântica é um dos ambientes mais ricos e ameaçados do mundo, o que deveria ter estimulado em muito o estudo e a conservação do Bioma, mas a fauna de Hymenoptera permanece ainda relativamente pouco conhecida. Em especial, a fauna de abelhas da floresta ombrófila densa é pouco estudada em comparação à fauna das áreas abertas brasileiras. O projeto temático "Biodiversidade de Hymenoptera e Isoptera: riqueza e diversidade ao longo de um gradiente latitudinal na Mata Atlântica - a floresta úmida do leste do Brasil", integrante do Programa Biota-Fapesp, foi idealizado com o objetivo de catalogar térmitas, formigas e famílias selecionadas de vespas ao longo da Mata Atlântica, disponibilizando dados que permitam melhor embasar a conservação deste bioma. O protocolo de amostragem aplicada para a coleta de himenópteros (excluindo as formigas) empregou armadilhas Malaise, pratos-armadilha de cor amarela e esforço similar na varredura de vegetação ao longo de 17 localidades selecionadas, representando um gradiente de quase 20° de latitude na Mata Atlântica, dos Estados da Paraíba até Santa Catarina. Este protocolo foi definido para otimizar a coleta de vespas, sendo as abelhas um produto secundário da amostragem (levantamentos de abelhas em geral utilizam captura em flores ao longo das estações do ano). No entanto, devido à escala regional do projeto e ao grande esforço de amostragem, uma expressiva quantidade de abelhas foi coletada durante o projeto, incluindo novos registros de abelhas para a Mata Atlântica. Foi amostrado um total de 797 espécimes distribuídos em 105 espécies de abelhas; o grupo de abelhas mais rico e abundante foi Meliponina. Uma análise de correspondência "destendenciada" ('detrended correspondence analysis') aplicada à uma matriz de presença ou ausência de Meliponina revela a relativamente fraca influência do gradiente latitudinal na composição das assembléias de abelhas de Mata Atlântica. Uma listagem das espécies amostradas por localidade, com suas freqüências relativas, é apresentada e discutida.

Differentiation of Melipona quadrifasciata L. (Hymenoptera, Apidae, Meliponini) subspecies using cytochrome b PCR-RFLP patterns

Melipona quadrifasciata quadrifasciata and M. quadrifasciata anthidioides are subspecies of M. quadrifasciata, a stingless bee species common in coastal Brazil. These subspecies are discriminated by the yellow stripe pattern of the abdominal tergites. We found Vsp I restriction patterns in the cytochrome b region closely associated to each subspecies in 155 M. quadrifasciata colonies of different geographical origin. This mitochondrial DNA molecular marker facilitates diagnosis of M. quadrifasciata subspecies matrilines and can be used to establish their natural distribution and identify hybrid colonies.

A assembléia de abelhas (Hymenoptera, Apidae) de uma área restrita de campos naturais do Parque Estadual de Vila Velha, Paraná e comparações com áreas de campos e cerrado

A assembléia de abelhas (Hymenoptera, Apidae) de uma área restrita de campos naturais do Parque Estadual de Vila Velha, Paraná e comparações com áreas de campos e cerrado. Foram realizadas coletas sistemáticas de abelhas em uma área restrita no Parque Estadual de Vila Velha, Paraná, no período de outubro de 2003 a setembro de 2004. Essa amostragem visou gerar subsídios para comparações entre áreas de campo dentro do Parque e com outras áreas de vegetação aberta brasileiras. A área de estudo possui cerca de quatro hectares e foi subdividida em quatro subáreas contínuas e com o mesmo perímetro. As abelhas em flores e em vôo foram capturadas com auxílio de rede entomológica. Foram coletados 1.459 espécimes pertencentes a 122 espécies de abelhas. Estas espécies estão distribuídas em 51 gêneros, 20 tribos e cinco subfamílias. As plantas visitadas correspondem a 93 espécies, pertencentes a 62 gêneros e 29 famílias. Neste estudo e em outro estudo realizado anteriormente em outra área do Parque foram coletadas 222 espécies sendo 74 espécies em comum as duas áreas e uma soma de 148 espécies restritas a uma ou outra área. Ainda, registros históricos de abelhas para Vila Velha elevam a riqueza conhecida para 261 espécies. O índice de similaridade de Morisita entre as áreas foi de 0,50 quando utilizadas as plantas como variáveis e 0,88 quando utilizados gêneros e espécies de abelhas, revelando uma notável heterogeneidade. Comparações entre as áreas do Parque Estadual de Vila Velha e áreas de campos e cerrado foram realizadas através do índice de similaridade e da análise de correspondência. Foram observados dois agrupamentos fortemente evidentes, referentes aos campos e ao cerrado. A análise de correspondência sugere que alguns gêneros podem ser relacionados a determinadas formações vegetais. Esse estudo indica que a fauna de abelhas de Vila Velha é composta principalmente por gêneros relacionados às áreas de campos, porém com presença de elementos de cerrado.

Description of the pupal case of Systropus (Systropus) nitidus Wiedemann, 1830 (Diptera, Bombyliidae, Toxophorinae, Systropodini)

The pupal case of Systropus (Systropus) nitidus Wiedemann reared from an unidentified tipical Limacodidae (Lepidoptera) cocoon is described and illustrated for the first time. Only species of Limacodidae are recorded as host of the immature stages of S. (Systropus). The geographical distribution of S. (Systropus) nitidus is restricted to Brazil, from Pará to Santa Catarina states. This is the first pupal case description and illustration of a Neotropical species of the subgenus Systropus.