Bees play an important ecological role worldwide since they pollinate numerous crops and wild plants and an economic role in agriculture. The pollination of such fruit and vegetable crops as apple, pear, melon, strawberry, onion, pea, asparagus, celery, cotton and alfalfa depend on populations of domestic bees or other native, wild, solitary, or social species (Ollerton et al., 2010; Koh et al., 2016). Bees in hive are continuously exposed to pathogens affecting their populations in winter, low temperatures, increased humidity, shortages of pollinic resources, hive movements and deficient health management carried out by beekeepers. These factors predispose bees to the action of pathogens and parasites that affect the colonies of such species as
Although the
The fungus spores are known to be viable for about fifteen years; they are transported in the pollen brought by the foraging bees to the colony, causing greater risk to the brood (Flores et al., 2005; Lopes et al., 2015). Furthermore, pollen bread and preserved pollen were reported to be reservoirs of entomopathogenic spores, which remain viable for months (Menapace, 1980; Hale Wang & Granados, 2000). Ascospores of Spanish strains remain viable in commercial pollen after two years of conservation (Tejerina et al., 2019). Larvae are the most susceptible between the fourth and fifth days of age, although some authors argue that this happens between the second and fourth days before the cells are sealed. Therefore, larvae must have consumed
This disease not only affects the larvae of worker bees but also those of drones and queens (Wynns, 2012). While it has not been proven to affect adult bees, they would transmit the disease among hives. Twenty-four hours after the larvae have ingested the ascospores, the first signs of the disease appear, starting with a reduction in the diet (food consumption). The larvae die within 48 h, and fungal mycelia become visible on the surface of the larvae after 72 h (Aronstein & Murray, 2010). Enzyme machinery involved in the virulence of
Although entomopathogenic strains have been registered in such countries as the United States, Spain, New Zealand, Australia and Canada (Albo & Reynaldi, 2010). They have been reported in southern Argentina (Reynaldi et al., 2003; Reynaldi et al., 2015) and the neighboring countries of Chile (Reynaldi et al., 2003), Brazil (Castagnino et al., 2006) and Uruguay (Rivas & Bettucci, 2007), but there have been no reports for northern Argentina. We investigated pollen from Argentinian provinces because it is available on the local market and many producers use commercial pollen to feed their bee colonies in winter.
The objective of this work was to isolate, identify, and characterize the
Commercial pollen was obtained dried in 2014, 2015 and 2016; one random sample from each province was acquired from herbalists from the Argentinian provinces of Buenos Aires, Corrientes, Entre Ríos, Jujuy, Misiones, San Luis, Santa Fe and Tucumán (Fig. 1). Although the treatment that each sample received is not known, the norms for commercialization proposed by the National Administration of Drugs, Food and Medical Technology had to be followed, which guarantees that Argentinian products are effective, safe and of good quality. The eight samples received from herbalists were stored at 20ºC in the dark until they were processed in the laboratory. Then, 1 g of each pollen sample was seeded on a selective medium containing agar malt and yeast extracts with 20% glucose (hereafter MY20) (Takatori & Tanaka, 1982) and incubated under microaerophilia conditions at 30±2°C for fourteen days.
The fungal strains were selected according to their macroscopic characteristics in the MY20 culture medium. The shape, size and color of the colony as well as the microscopic features, namely shape and size of sporocysts, asci, and ascospores (µm) were taken into account. The samples were prepared with lactophenol blue and observed at 40X and 100X under a Zeiss microscope; 1000 sporoscysts and the average size of asci and ascospores were measured (µm).
Genomic DNA was obtained from the collected samples, using the mycelium grown in MY20 liquid medium. The mycelium was washed twice with 1 mL of cold solution (Tris-HCl 0.1M, EDTA 0.02M) and spun at 12,000 rpm for 3 min. Subsequently, the mycelium was crushed with a sterile glass rod in the presence of 1 mL of extraction buffer (Tris-HCl 100mM pH 8, NaCl 1.5M, EDTA 50mM pH 8, Proteinase K, 0.1mg/ml, β-mercaptoethanol 10 mM, SDS 2%) and digested at 60ºC by agitation with vortex every 10 min. The DNA was purified from the supernatant of this digestion using chloroform:isoamyl alcohol (24:1) twice and potassium acetate 3M only once. The DNA was subsequently precipitated with 100% isopropanol, 70% cold ethanol wash, spun at 12,000 rpm for 5 min, dried at room temperature, resuspended in distilled nuclease-free water and stored at −20ºC until use. Genomic DNA was revealed in agarose gel at 1%, with 3 μL of ethidium bromide (0.008 mg/μL) for every 75 mL of agarose solution.
To characterize the fungal isolates, the ITS1-5.8S-ITS2 region of the ribosomal DNA was amplified using the primers described by White et al. (1990), ITS1 5′-TCCGTAGGTGAACCTGCGG and ITS4 5′-TCCTCCGCTTATTGATATGC. The PCR was carried out in 20 μl of the final volume containing 1X buffer, magnesium chloride 2.5 mM, dNTPs 200 µM, 10ρM of each primer and 0.5 U Taq polymerase, using a cycle composed of an initial denaturation for 4 min at 94ºC and thirty-five cycles of 40 s at 94ºC, 40 s at 52ºC, and 40 s at 72ºC, with a final extension of 10 min at 72ºC.
The PCR products were checked on agarose gels at 2%, sequenced using the Macrogen Korea service and analyzed with the MEGA6 program. The sequences were contrasted against the database of the NCBI (
All sequences were analyzed using BioEdit and Clustal W before tree construction. A phylogenetic analysis was carried out using the program TNT (tree analysis using New Technology), which performs phylogenetic analysis of parsimony (Goloboff et al., 1999). The gaps (indels) were treated as a fifth state since they represented insertion-deletion events. The analysis included twenty-four sequences of the genus
Argentinian samples of corbicular pollen were disintegrated and homogenized with a mortar and 0.5 g of the total was taken for additional treatment with acetolysis (Erdtman, 1960). The pellet was glycerin mounted and paraffin sealed. Observations were made with a Boeco optical microscope. To determine frequency classes, 600 pollen grains were counted (Louveaux et al., 1978). Pollen types were compared and identified to the palynological deposits from the palynology laboratory of the Faculty of Agrarian Sciences, National University of Jujuy, and by consulting the palynological atlas.
The bees used to carry out the assays belong to the hybrid subspecies
We used three blind cores (no initiating queen), that had been prepared in the spring of 2016 and contained a standard open breeding frame, a breeding frame operculated with nurse bees and a frame with honey, as proposed by Ahmed (2008). We waited until the birth and posture of the queen. When the nuclei grew and consolidated, with about 10,000 bees per frame, they were transferred to three standard brood boxes and allowed to grow again until we obtained a population of approximately 50,000 bees, as proposed by Audisio & Benítez-Ahrendts (2011). Above this brood nest, we placed a medium super frame (without a queen excluder, to allow the queen to pass), which contained twenty-four experimental frames specially designed for assays, measuring 20×10 cm. When the worker bees stretched the beeswax, the posture of the queen was observed, and then when the first bees were born, the state of the hives was monitored monthly. Trials were conducted in the late spring of 2017 and 2018 when temperatures were below 20ºC, the morning temperature registered in this season in Jujuy province. The three hives used for the study were eliminated at the end of the trial to prevent the spread of the disease.
Portions of each fungal strain inoculated with agar and mycelium (about 36 mm2) were placed on Petri dishes in the middle of the MY20 culture and the fungi were grown for ten days until the maturation of sporocysts, under microaerophilia conditions at 30±1ºC. Microscopic observation was performed to confirm the presence of mature spores with a 40 X objective.
An aliquot was taken from each
Before being administered to the hives, the ascospore suspensions were spun at 4000 rpm for 5 min and the cell pellets were resuspended in 5 mL of sterile water and honey (1:1). For the control groups (without ascospores), a suspension of 5 mL of sterile water and honey was used (1:1).
The brood chambers of the three assayed colonies were checked before the administration of the ascospores and showed no symptoms or signs of the disease. The uniform posture of the queen and the presence of larvae were verified. Ten open breeding trial frames containing 5- to 6-day-old larvae were selected for the inoculation of the fungal strains. Two repetitions per area of 20×10 cm were performed and areas with capped and uncapped cells were counted before the administration of the strains.
Subsequently, all larvae were fed using a sterile 2.5 mL syringe. They were divided into three groups; the first two were administered a drop of approximately 5 μL of each ascospore suspension at different concentrations: at the minimum concentration and the maximum concentration, respectively, while the third group was used as control (food without ascospores) (See Tab. 3). After feeding, the frames were taken to the original hive so that the cells could be covered by worker bees. At 48 h, the frames were removed from the apiary and incubated in the laboratory for ten days in hives designed for the frames. The mummified larvae in each cell were then counted. To replicate the humidity and temperature environmental conditions in the laboratory, we used a stove at 30±1ºC with 60% humidity.
Data on the number of infected larvae were expressed as mean ± standard deviation of each assay. Statistical analysis of results was performed using ANOVA and Tukey’s mean comparison test, with a 0.5 probability of making a type I error, using Infostat as the statistical package (Di Rienzo et al., 2008).
Three strains of the genus
Macroscopic description of isolated
strains | Origins | Sporocyst diameter (µm) | Ascus diameter average (µm) | Ascospores length average (µm) | |
---|---|---|---|---|---|
PMis | Misiones | 60–75 | 9 | 1–2 | |
PJuy | Jujuy | 65–70 | 9 | 1–2 | |
PER | Entre Ríos | 60–70 | 8 | 1–2 |
The
In addition, when the botanical origin of each pollen sample was identified, interestingly, the predominant pollen belonged to the genus
Botanical origin associated with the three strains of
Pollen types | Porcentage by provincie | ||
---|---|---|---|
Entre Ríos | Jujuy | Misiones | |
Amaranthaceae | 8.87 | 6.42 | 12.05 |
Apiaceae | 0.55 | 0.28 | 0 |
Caesalpiniaceae | 0.92 | 0 | 0 |
1.11 | 0 | 0.17 | |
Fabaceae | 0 | 1.4 | 0 |
14.23 | 0 | 18.59 | |
0 | 0.56 | 0 | |
Monocotyledoneae | 0.74 | 0 | 1.55 |
Myrtaceae | 0 | 97.6 | 0 |
0 | 1.96 | 0 | |
Poaceae | 0 | 0 | 0.34 |
2.22 | 1.4 | 1.38 | |
0 | 5.31 | 0 | |
68.58 | 73.18 | 61.96 | |
0 | 0 | 0.34 | |
1.66 | 3.07 | 1.72 | |
0 | 3.91 | 0 | |
Solanaceae | 0.55 | 1.12 | 0.69 |
0.55 | 1.4 | 0.52 |
A total of 872bp were aligned, 299 sites were considered monomorphic, 504 sites were considered polymorphic or phylogenetically informative segregants, and 359 polymorphic sites had more than two variants. The parsimony analysis resulted in two parsimonious trees, with 1889 steps.
The
The minimum concentration of ascospores administered in sugar syrup did not differ significantly (ANOVA, p=0.83) in the number of mummified larvae. No mummified larvae were detected in connection with the Entre Rios or Misiones strains, and only the Jujuy strain showed the presence of 1±1 mummified larva (Tab. 3). In contrast, when the suspension with the maximum concentration of ascospores was administered to the larvae, the registered number of mummified larvae with each of the fungal strains was higher (Misiones 30±7, Entre Rios 10±1 and Jujuy 23±3) than the number of mummified larvae fed the minimum concentration of ascospores (Tab. 3). The number of mummified larvae with high concentrations of ascospores exhibited significant differences between the pathogenic effects of the strains from each province (ANOVA, p<0.05) (Fig. 5). The
Number of larvae mummies with
Strains | Number of mummies | Number of non-mummified larvae (Witness) | Number of infected larvae (5×103 spores/mL) | Number of infected larvae (5×106 spores/mL) | Total number of capped cells | ||
---|---|---|---|---|---|---|---|
(5×103 spores/mL) | (5×106 spores/mL) | Without sporocyst | |||||
Misiones | 0 | 30±7 | 3±3 | 100±40 | 138±1 | 127±29 | 43±6 |
Entre Ríos | 0 | 10±1 | 2±2 | 112±2 | 128±12 | 83±9 | 28±11 |
Jujuy | 1±1 | 23±3 | 5±1 | 102±32 | 134±10 | 85±43 | 54±8 |
The larvae inoculated with
In this study, we isolated strains belonging to the genus
Commercial pollen subjected to different treatments after harvesting, in which it is dried at temperatures below 40–45ºC, eliminates excess humidity (6 to 7%) and preserves the bromatological characteristics required for its commercialization (Aranda-Escribano et al., 1999; Baldi-Coronel et al., 2004). Although these treatments eliminate most of the microorganisms, an incorrect procedure could transport diseases to the bee colonies, as demonstrated by Graystock et al. (2016), who recorded the presence of
The morphological observation indicated that the size of the strains’ sporocysts and the ascospores were similar to those reported in other works from both southern Argentina and other parts of the world (Albo & Reynaldi, 2010; Aronstein & Murray, 2010; Wynns et al., 2013; Chen et al., 2018; Pereira et al., 2019). The phylogenetic analysis allowed us to establish relationships between strains found in some countries and isolates of commercial pollen from Argentina. The
Although multifloral pollen is known to contain ascospores (Pereira et al., 2019), the palynological origin associated with its transmission has not been determined. In this work, botanical determinations of pollen grains from Argentinian provinces are reported to corroborate their provenance. In addition, the most representative percentages of each floral resource used by
Our pathogenicity assays of the different strains in bee larvae allowed us to assess their potential to infect and mummify larvae of local bees that have never been exposed to this entomopathogen. Flores et al. (2004) evaluated the percentage of mummified larvae that were stressed at 18ºC for 24 h. These larvae came from hives which were treated with water spray with ascospores, pollen with ascospores or sugar syrup containing the ascospores; the third technique was the least effective in achieving a pathogenic effect. These assays were performed with a concentration of 1,250 spores/hives. In this work, a concentration of 5×106 spores/mL was used and the percentage of mummies was lower than that found by Flores et al. (2004) and Palacios et al. (2007), when they used sugar syrup. Both authors warned that the percentage of mummification depends on the stress conditions of the larvae when they receive the ascospores or on their susceptibility to contract the ectoparasite according to the period of the year.
The administration of the minimum concentration of ascospores to the larvae did not result in the disease. Jensen et al. (2013) indicated that the minimum concentration for successful infection in healthy hosts should be 5×105 ascospores/mL, which could lead to the death of the population. The pathogenicity also depends on the origin of the inoculum with ascospores. Flores et al. (2004) performed the infection techniques with ascospores from macerated bee mummies, while Palacios et al. (2007) maintained ascospores on integral rice kernels (IRK) medium and MY20. The lower percentages of infection of the larvae achieved by the direct method of feeding than those obtained in other works could be attributed to the ascospores coming from the cultivation of commercial pollen.
In addition, this low percentage of mummified larvae in comparison to other studies can also be attributed to the genetic characteristics of the bee colonies kept in Jujuy, since hybrids of Africanized bees belonging to the subspecies of
Strains of