Natalia Damiani

Nosema ceranae development in Apis mellifera: influence of diet and infective inoculum

Summary To investigate the effect of nutritional condition of the honey bee Apis mellifera on the development of the microsporidian parasite Nosema ceranae under laboratory conditions, newly emerged bees were confined and fed on three ad libitum diets: high fructose corn syrup (HFCS) + fresh bee bread; HFCS + a commercial mixture of amino acid and vitamin, and HFCS. On day 7 post-emergence, bees from each diet treatment were individually infected with 4.60 × 104, 2.30 × 105 or 1.15 × 106 spores of N. ceranae, keeping later on the same diet. On days 3, 6, 9, and 12, post-infection bee midguts were removed to individually quantify the spores developed. The results indicate that this parasite multiplies successfully regardless of the inoculum given or the nutritional status of its host. When bees are fed on pollen, however, the parasite develops quickly, exhibiting significantly higher intensities than under other treatments. The longevity of infected bees fed on the same diet was not affected by the degree of parasitism, but by the quality of the ad libitum diet administered. The data demonstrate a parasite development that depends on host-condition. This should be considered when designing experiments to evaluate the development and virulence of this pathogen.

Repellent and acaricidal effects of botanical extracts on Varroa destructor

Extracts of indigenous plants from South America have shown a broad spectrum of bioactivities. No-contaminant and natural substances have recently resurged as control treatment options for varroosis in honey bee colonies from Argentina. The aim of this work was to evaluate the biological activity of botanical extracts from Baccharis flabellata and Minthostachys verticillata on Varroa destructor and Apis mellifera. The acaricidal and insecticidal activities were assessed by the spraying application method. Both ethanolic extracts showed high levels of toxicity against the mites and were harmless to their host, A. mellifera. During the attractive-repellent test, the olfactory stimulus evoked for the extract from B. flabellata resulted as a repellent for mites. The aromatic stimulus of these extracts would be strong enough to cause disturbance on the behavior of V. destructor. Thus, the repellent effect of these substances plus the toxicity on mites postulate these botanical extracts like promising natural compound to be incorporated for the control of varroosis.

Effect of Propolis Oral Intake on Physiological Condition of Young Worker Honey Bees, Apis Mellifera L.

Abstract Honey bees collect resin from various plant species and transform it into propolis that is incorporated into the nest. The role of resins in the bee health field is poorly understood. The aim was to evaluate the effects of forced consumption of propolis on the physiological condition and short-term survival of Apis mellifera worker bees. It was tested if the number of circulating hemocytes in hemolymph, the abdominal fat bodies and the hypopharyngeal glands development were affected by the feeding with propolis extracts in laboratory conditions during the warm and the cold seasons. Propolis added to sugar candy was consumed by workers for fourteen days without affecting the bee survival. The number of circulating hemocytes in hemolymph remained constant despite the differential diet during the experiment. However, the development of fat bodies and hypopharyngeal glands was altered by propolis ingestion. The abdominal fat body development in winter bees diminished after fourteen days of propolis consumption, while it increased in summer bees. The hypopharyngeal gland development decreased for the assayed period in workers from both seasons. Our results encourage us to continue exploring this research field and learn how long-term forced ingestion of a plant-derived compound, a non-nutritive substance, can modify physiological bee parameters. A broader understanding of the multiple roles of propolis in the health of the honey bee colonies could be obtained by studying the ways in which it is processed and metabolized and the effect that generates in another physiological responses.

A scientific note on the first report of honeybee venom inhibiting Paenibacillus larvae growth

In the eusocial honeybee, Apis mellifera, worker bees use a stinging apparatus for defense. The sting is supplied with venom by glands localized in the abdomen. Honeybee venom (BV) is composed of at least 18 bioactive molecules, ranging from biogenic amines to proteins whose structure and function have been largely determined. These include peptides such as melittin, apamin, adolapin, and mast cell degranulating peptide; biologically active amines; enzymes as phospholipase A2 (PLA2); and a few nonpeptide components (Peiren et al. 2005; Matysiak et al. 2011). Melittin and PLA2 are the most abundant proteins, representing 50 and 12 % of BV dry weight, respectively. Furthermore, the venom gland has been recently reported as an important source of antimicrobial substances with proven antibacterial and antifungal action (Yu et al. 2012; Han et al. 2013). Nevertheless, data about the effects of BV on infectious pathogens of honeybees are previously absent from the literature. In this study, we proved that BV is able to inhibit the growth of a pathogenic bacterium affecting honeybee colonies. Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most destructive brood diseases of A. mellifera. Traditional control by chemotherapy has caused residues in hive products, and, more disturbingly, resistant pathogens (Evans 2003). These facts have driven studies on natural alternatives for AFB control. Thus, the aim was to analyze the effects of honeybee venom on P. larvae growth as well as its effect on adult bee survival after chronic exposure to BV. P. larvae strains had been isolated from larvae with AFB from four apiaries in Argentina and PL33 (UB-CIDEFI) like reference strain. All strains were genotypically identified using PL2-Fw and PL2-Rv primers, and characterized like ERIC I with ERIC1R -and ERIC2 primers (Genersch et al. 2006). For BV extraction, A. mellifera forager bees were obtained by covering the hive entrance and collecting them in their back flight. Venom sacs were dissected by pulling the sting apparatus from 30 bees. The resultant droplet of BV from the tip of the sting was placed on sterile slides, allowed to dry in dark for 24 h, were collected by scraping and then weighed. P. larvae isolates were exposed to serial dilutions of BV. One hundred microliters of Mueller–Hinton broth, yeast extract, and thiamine broth (Gende et al. 2008) were placed per well. BV stock solution and 50 μL of the bacterial suspension (0.5 McFarland scale) were added to each well (serial dilutions from 200 to 1.56 μg/mL). Incubation took place at 37oC for 48 h. The inhibition of bacterial growth was confirmed by resazurin method (Damiani et al. 2014). Serial dilutions from 100 to 0.04 μg/mL of oxytetracycline (Fluka Analytical) were included as a positive control (Alippi et al. 2007). The lowest concentration of each substance that showed inhibition was considered as the minimal inhibitory concentration (MIC). The minimal bactericidal concentration (MBC) was estimated transferring 100 μL honeybee venom/ Paenibacillus larvae / antimicrobial activity / Apismellifera

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 ± 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.