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Using palynological evidence from royal jelly to mediate the spread of Paenibacillus larvae in Brazil

Usando evidências palinológicas em geléia real para intervir na disseminação de Paenibacillus larvae no Brasil

ABSTRACT

Royal jelly may contain pollen grains and their presence can be used to determine the phytogeographical origin of the product. This study analyzed the phytogeographical origin of commercial royal jelly samples from São Paulo State, tested as part of the Brazilian Federal Inspection System (SIF), found to be contaminated with spores of the bacterium Paenibacillus larvae, that causes the American Foulbrood Disease. The pollen grains of Castanea had the highest total percentage, with lower percentages of Cirsium/Carduus, Cistus, Parthenocissus, Prunus, Quercus, Robinia, Scrophulariaceae, Taraxacum, Tilia, among others. This pollen spectrum is incompatible with royal jelly samples produced in Brazil. The pollen spectrum resembled that of an imported product, compatible with the Northern Hemisphere origin. Brazilian legislation does not require the phytogeographic origin of imported bee products to be analyzed by palynological procedures, but it is mandatory to have a certificate issued by the country of origin attesting the absence of pathogens, monitored with the objective of preventing the exotic diseases from entering Brazil. Palynology, therefore, proved to be fundamental in detecting imports of this contaminated batches.

Keywords:
Apis mellifera; Forensic palynology; foulbrood disease; Melissopalynology; pollen analysis

RESUMO

A geléia real pode conter grãos de pólen e sua presença pode ser usada para determinar a origem fitogeográfica do produto. Este estudo analisou a origem fitogeográfica de amostras comerciais de geléia real do Estado de São Paulo, com registro no Sistema de Inspeção Federal (SIF) brasileiro, que estavam contaminadas com os esporos da bactéria Paenibacillus larvae, causadora da doença Cria Pútrida Americana. Os grãos de pólen de Castanea apresentaram a maior porcentagem total, com menores percentuais de Cirsium/Carduus, Cistus, Parthenocissus, Prunus, Quercus, Robinia, Scrophulariaceae, Taraxacum, Tilia, dentre outros. Este espectro polínico é incompatível com amostras de geléia real produzidas no Brasil. O espectro polínico assemelhou- se ao de um produto importado, compatível com a origem do Hemisfério Norte. A legislação brasileira não exige que a origem fitogeográfica dos produtos apícolas importados seja analisada por procedimentos palinológicos, mas é obrigatório ter um certificado atestando a ausência de patógenos emitido pelo país de origem, monitorado com o objetivo de impedir que doenças exóticas penetrem no Brasil. A palinologia, portanto, provou ser fundamental na detecção de importações desses lotes contaminados.

Palavras-chave:
Análise polínica; Apis mellifera; cria pútrida americana; Melissopalinologia; Palinologia forense

Introduction

A decline in the honey bee population has been reported in various countries over the past decade by the FAO (United Nations Food and Agriculture Organization) and the NAS (U.S. National Academy of Sciences), including populations in Brazil. This is a matter of great concern due to the decline in pollination of important food crops (Klein et al. 2007Klein, A.M., Vaissière, B.E., Cane, J.H., Steffan-Dewenter, I., Cunningham, S.A., Kremen, C. & Tscharntke, T. 2007. Importance of pollinators in changing landscapes for world crops. Proceedings of the Royal Society B: Biological Sciences 274: 303-313.). The honey bee mortality observed is not seasonal and there seems to be a complex interaction between various factors, including insecticide application by farmers as well as fragmentation and degradation of habitats (Johnson 2010Johnson, R. 2010. Honey Bee Colony Collapse Disorder. Available in http://www.fas.org/sgp/crs/misc/RL33938.pdf. (access in 14-III-2018).
http://www.fas.org/sgp/crs/misc/RL33938....
). However, the data are still imprecise and the causes are undefined due to the absence of standards and/or weak monitoring (Pires et al. 2016Pires, C.S.S., Pereira, F.M., Lopes, M.T.R., Nocelli, R.C.F., Malaspina, O., Pettis, J. & Teixeira, E.W. 2016. Enfraquecimento e perda de colônias de abelhas no Brasil: há casos de CCD? Pesquisa Agropecuária Brasileira 51: 422-442.).

Although honey bees are attacked by many parasites and pathogens (Chen et al. 2004Chen, Y., Zhao, Y., Hammond, J., Hsu, H., Evans, J.D. & Feldlaufer, M. 2004. Multiple virus infections in the honey bee and genome divergence of honey bee virus. Journal of Invertebrate Pathology 87: 84-93.), Africanized bees are more tolerant and resistant to several pathogens. However, in recent years there has been a sudden weakening of swarms in various places in the southeast of Brazil, with mortality of adult bees and the appearance of abnormal symptoms in broods (Message et al. 2012Message, D., Teixeira, E.W. & De Jong, D. 2012. Situação da sanidade das abelhas no Brasil. In: V.L. Imperatriz-Fonseca, D.A.L. Canhos, D.A. Alves & A.M. Saraiva (orgs.). Polinizadores no Brasil: Contribuição e perspectivas para a biodiversidade, uso sustentável, conservação e serviços ambientais. EDUSP, São Paulo, pp. 237-256.).

American foulbrood disease, caused by the bacterium Paenibacillus larvae, one of the main cause of losses of colonies in the world (Funfhaus et al. 2013Funfhaus, A., Poppinga, L. & Genersch, E. 2013. Identification and characterization of two novel toxins expressed by the lethal honeybee pathogen Paenibacillus larvae, the causative agent of American foulbrood. Environmental Microbiology 15: 2951-2965.). This pathogen contaminates colonies through spore ingestion by larvae (Garrido-Bailón et al. 2013Garrido-Bailón, E., Higes, M. & Martínez-Salvador, A. 2013. The prevalence of the honeybee brood pathogens Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius in Spanish apiaries determined with a new multiplex PCR assay. Microbial Biotechnology 6: 731-739.), causing their death, consequently a decrease in the colony population. The bacterium P. larvae is exotic in Brazil and American foulbrood disease was only detected twice in the country. The disease was detected for the first time in 2001 in the municipality of Candelária, Rio Grande do Sul State, where samples of adult bees, honey, pollen and honeycombs were collected and analyzed (Schuch et al. 2003Schuch, D.M.T., Tochetto, L.G. & Sattler, A. 2003. Isolamento de esporos de Paenibacillus larvae subsp. larvae no Brasil. Pesquisa Agropecuária Brasileira 38: 441-444.). Spores of the bacterium were detected in three groups of adult bees and in honeycombs, as well as in imported honey and pollen samples. In 2006, clinical signs of the disease were reported in samples from Quatro Barras, Paraná State. This bacterium was quickly controlled and extinguished in the country (MAPA 2006MAPA. 2006. Nota técnica DSA nº52/2006. Ocorrência de “Cria Pútrida Americana” no município de Quatro Barras, estado do Paraná-Brasil. Ministry of Agriculture of Brasil, Brasília.).

Exotic diseases can enter new territories by natural migration of bees, or by importing queen bees or bee products contaminated with pathogens (Hitchcock & Revell 1963Hitchcock, J.D. & Revell, I.L. 1963. The spread of American foulbrood from pollen trapped from bees’ legs. American Bee Journal 103: 220-221., Gochnauer & Corner 1974Gochnauer, T.A. & Corner, J. 1974. Detection and identification of Bacillus larvae in a commercial pollen sample. Journal of Apicultural Research 13: 264-267., Hale & Menapace 1980Hale, P.J. & Menapace, D.M. 1980. Effect of time and temperature on the viability of Ascosphaera apis. Journal of Invertebrate Pathology 36: 429-430., Flores et al. 2005Flores, J.M., Spivak, M. & Gutiérrez, I. 2005. Spores of Ascosphaera apis contained in wax foundation can infect honeybee brood. Veterinary Microbiology 108: 41-144., Higes et al. 2008Higes, M., Martín-Hernández, R., Garrido-Bailón, E., García-Palencia, P. & Meana, A. 2008. Detection of infective Nosema ceranae (Microsporidia) spores in corbicular pollen of forager honeybees. Journal of Invertebrate Pathology 97: 76-78., Pettis et al. 2013Pettis, J.S., Lichtenberg, E.M., Andree, M., Stitzinger, J., Rose, R. & Vanengelsdorp, D. 2013. Crop pollination exposes honey bees to pesticides which alter their susceptibility to the gut pathogen Nosema ceranae. PLoS One 8: e70182., Fries et al. 2013Fries, I., Chauzat, M.P., Chen, Y.P., Doublet, V., Genersch, E., Gisder, S., Higes, M., McMahon, D.P., Martín-Hernández, R., Natsopoulou, M., Paxton, R.J., Tanner, G., Webster, T.C. & Williams, G.R. 2013. Standart methods for Nosema research. Journal of Apicultural Research 52: 1-28., OIE 2014OIE - World Organization of Animal Health. 2014. American foulbrood of honeybees. In: Manual of diagnostic tests and vaccines for terrestrial animals. v. 1, section 2.2, Chapter 2.2.2. Available in http://www.oie.int/fileadmin/Home/fr/Health_standards/tahm/2.02.02_AMERICAN_FOULBROOD.pdf (access in 18-III-2018).
http://www.oie.int/fileadmin/Home/fr/Hea...
, Guimarães-Cestaro et al. 2016Guimarães-Cestaro, L., Serrão, J.E., Message, D., Martins, M.F. & Teixeira, E.W. 2016. Simultaneous detection of Nosema spp., Ascosphaera apis and Paenibacillus larvae in honeybee products. Journal of Hymenoptera Research 49: 43-50.). Diseases caused by microorganisms are of particular concern due to the easy transmission of resistant spores, which can remain viable for decades (Hornitzky 2010Hornitzky, M. 2010. Honey bee diseases. Australia and New Zealand Standard Diagnostic Procedure. Available in http://www.agriculture.gov.au/animal/health/laboratories/procedures/anzsdp/honey-bee-diseases/ (access in 27-III-2018).
http://www.agriculture.gov.au/animal/hea...
). For example, spores of P. larvae can remain viable for about 70 years in the environment (Shimanuki & Knox 1994Shimanuki, H. & Knox, D.A. 1994. Susceptibility of Bacillus larvae to Terramycin. American Bee Journal 134: 125-126., 2000).

Inside the bee colony, products such as honey, pollen, and royal jelly are susceptible to contamination by these pathogens, mainly by being stored in comb cells by the bees and transferred through trophallaxis (OIE 2014OIE - World Organization of Animal Health. 2014. American foulbrood of honeybees. In: Manual of diagnostic tests and vaccines for terrestrial animals. v. 1, section 2.2, Chapter 2.2.2. Available in http://www.oie.int/fileadmin/Home/fr/Health_standards/tahm/2.02.02_AMERICAN_FOULBROOD.pdf (access in 18-III-2018).
http://www.oie.int/fileadmin/Home/fr/Hea...
). Due to the high risk of introducing exotic pathogens into the country by imported bees’ products and queens, sanitary control is regulated by a specific resolution of the Brazilian Ministry of Agriculture. Among the requirements are documents from the country of origin that include the results of diagnostic tests.

Royal jelly is a product secreted by the hypopharyngeal glands of worker honeybees (Apis mellifera). It is derived mostly from the nutrients contained in the pollen grains ingested by the bees. Pollen is the main source of proteins, peptides and secondary metabolites in royal jelly. Therefore, pollen is the main precursor product that results in royal jelly and substantially influences its biological properties (Stocker 2005Stocker, A. 2005. Isolation and characterisation of substances from Royal Jelly. Thèse. Université d’Orléans, Université Technique de Munich. Available in https://tel.archives-ouvertes.fr/tel-00008586. (access in 14-III-2018).
https://tel.archives-ouvertes.fr/tel-000...
). Although it does not make sense to refer to the botanical origin of royal jelly, because it is a bee secretion, observing the pollen types and their percentages (pollen spectra) can provide some evidence regarding the phytogeographical origin of a royal jelly sample. Pollen flow into the product is an accidental process, introduced from the internal environment of the colony, during manipulation in the mouthparts of the adult bees when feeding queen larvae or adult queens. As the royal jelly is not stored in the hive, it can be found pollen grains that happened to be in the bees' bodies or in their nectariferous vesicles (Simpson 1955Simpson, J. 1955. The significance of the presence of pollen in the food of worker larvae of honeybees. Quarterly Journal of Microscopical Science Archives 96: 117-120., Renner et al. 2003Renner, P., Hrassnigg, N. & Crailsheim, K. 2003. Trophallaxis between nurse bees and pollen foragers under laboratory conditions in Apis mellifera carnica. Apidologie 34: 492., Barth 2005Barth, O.M. 2005. Botanical resources used by Apis mellifera determined by pollen analysis of royal jelly in Minas Gerais, Brazil. Journal of Apicultural Research 44: 78-81.).

Pollen grains analysis in bee products is an effective tool to support the certification of their phytogeographical origin because it shows the presence of pollen types that are characteristic of specific plants. However, according to Brazilian legislation, this analysis is not mandatory for imported or local bee products.

There are few palynological studies of royal jelly (Chauvin & Louveaux 1956Chauvin, R. & Louveaux, J. 1956, Etude macroscopique et microscopique de la gelée royale. L’apiculteur 1956: 33-43., Ricciardelli D’Albore et al. 1977Ricciardelli D’Albore, G., Battaglini, M. & D’Ambrosio, M. 1977. Considerazioni sui pollini della gelatina reale. Annali dell’Istituto Sperimentale per la Zoologia Agraria 5: 203-213., Ricciardelli-D’Albore & Battaglini 1978Ricciardelli-D’Albore, G. & Battaglini, M. 1978. Origine géographique de la gelée royale. Apidologie 9: 1-17., Biondi et al. 2003Biondi, C., Bedini, G. & Felicioli, A. 2003, Gelatina reale: metodologia proposta per la determinazione dell’origine geografica e della qualità. Apitalia 6: 32-37., Piana et al. 2006Piana, M.L., Belligoli, P., Persano Oddo, L. & Piperno, S. 2006. Pollen analysis of royal jelly: contribution to Analytical methods and characterization. Apiacta 41: 28-43., Dimou et al. 2007Dimou, M., Goras, G. & Thrasyvoulou, A. 2007. Pollen analysis as a means to determine the geographical origin of royal jelly. Grana 46: 118-122., Dimou et al. 2013Dimou, M., Tananaki, C., Goras, G., Karazafiris, E. & Thrasyvoulou, A. 2013. Melissopalynological analysis of royal jelly from Greece. Grana 52: 106-112.). In Brazil, only the studies carried out by (Barth 2005Barth, O.M. 2005. Botanical resources used by Apis mellifera determined by pollen analysis of royal jelly in Minas Gerais, Brazil. Journal of Apicultural Research 44: 78-81.) and (Morgado & Barth 2011Morgado, L. & Barth, O.M. 2011. The detection of pollen in royal jelly of honey bees (Apis mellifera). Journal of ApiProduct and ApiMedical Science 3: 137-139.) can be found, which samples are rich in pollen grains of Asteraceae, Cecropia, Eucalyptus, Melastomataceae and Mimosa pudica/M. scabrella.

This study evaluated the phytogeographical origin of royal jelly samples sold in São Paulo State, Brazil, tested by the Federal Inspection System (Sistema de Inspeção Federal - SIF) in which positive signs of Paenibacillus larvae were previously detected based on PCR results. The results of these analyses are useful for elucidating the possible dispersion of this pathogen in Brazil through the sale and distribution of bee products.

Material and methods

Eight royal jelly samples, which were inspected by the S.I.F., were analyzed using palynological techniques. They were purchased in São Paulo State, Brazil and were bottled from 2012 to 2014. Positive signs of Paenibacillus larvae were detected according to molecular analyses (Guimarães-Cestaro et al. 2016Guimarães-Cestaro, L., Serrão, J.E., Message, D., Martins, M.F. & Teixeira, E.W. 2016. Simultaneous detection of Nosema spp., Ascosphaera apis and Paenibacillus larvae in honeybee products. Journal of Hymenoptera Research 49: 43-50., Teixeira et al. 2018Teixeira, E.W., Guimarães-Cestaro, L., Marques, M.L.T., Alves, F., Message, D., Martins, M.F., Luz, C.F.P. & Serrão, J.E. 2018. Spores of Paenibacillus larvae, Ascosphaera apis, Nosema ceranae and Nosema apis in bee products supervised by the Brazilian Federal Inspection Service. Revista Brasileira de Entomologia (in press).).

The palynological treatment of the royal jelly followed Barth (2005)Barth, O.M. 2005. Botanical resources used by Apis mellifera determined by pollen analysis of royal jelly in Minas Gerais, Brazil. Journal of Apicultural Research 44: 78-81. using 1 g of the product for the analysis and preparation of the microscope slides which include sample dilution, centrifugation, resuspension in acetic acid for overnight dehydration, and subsequent acetolysis (Erdtman 1952Erdtman, G. 1952. Pollen morphology and plant taxonomy. Angiosperms (An introduction to Palynology. I). Almquist & Wiksell, Stockholm.). Three slides of each sample were prepared using standard uncolored and fuchsine stain colored (one slide) glycerine jelly (Barth 2005Barth, O.M. 2005. Botanical resources used by Apis mellifera determined by pollen analysis of royal jelly in Minas Gerais, Brazil. Journal of Apicultural Research 44: 78-81.). Fuchsin stained pollen grains were not photographed.

The identification of the pollen types was based on the European and Asian pollen flora catalogs (Punt & Clarke 1974Punt, W. & Clarke, G.C.S. 1974. The Northwest European Pollen Flora. Review of Palaeobotany and Palynology. Elsevier, New York., Lieux 1975Lieux, M.H. 1975. Dominant pollen types recovered from commercial Louisiana honeys. Economic Botany 29: 87-96.-1977Lieux, M.H. 1977. Secondary Pollen Types Characteristic of Louisiana Honeys. Economic Botany 31: 111-119.-1978Lieux, M.H. 1978. Minor honeybee plants of Louisiana indicated by pollen analysis. Economic Botany 32: 418-432., Göschl 2008Göschl, W. 2008. Beiträge zur Pollenmorphologie ausgewählter rezenter und fossiler Vertreter der Davidiaceae und Nyssaceae. Tese, Magister der Naturwissenschaften, Universität Wien, Viena., Haiqing 2015Haiqing, X. 2015. Common Cultivated Pollen Flora of China. China Scientific Book Services. Beijing, China.), among many publications that were consulted and that are part of the collection from the Palynological Research Centre (NPP) at the Institute of Botany, as well as others obtained from the Internet. The “Northwest European Pollen Flora” (Punt & Clarke 1974Punt, W. & Clarke, G.C.S. 1974. The Northwest European Pollen Flora. Review of Palaeobotany and Palynology. Elsevier, New York.) is the world´s most detailed pollen morphological study and covers around 130 families and ca. 600 species.

In the present study was adopted as counting methodology the observation of all pollen grains contained in the three slides. Pollen grains were identified as “pollen types” meaning that a single plant species, group of species or higher taxa could present with similar pollen morphologies.

In order to evaluate the phytogeographical origin of each sample, the percentage calculation representing all the pollen types per sample (Total Pollen Sum) was used. The pollen types were grouped according to their relative frequencies, following (Louveaux et al. 1978Louveaux, J., Maurizio, A. & Vorwohl, G. 1978. Methods of Melissopalynology. Bee World 59: 139-157.), corresponding to: Predominant pollen (P) = >45% of the total pollen grains counted; Secondary pollen (S) = 16% to 45%; Important Minor Pollen (I) = 3% to 15% and, Minor pollen (M) = <3%.

Although quantification by Lycopodium tracer spores provides a context for pollen spectra that is both useful for supporting phytogeographic origins of bee products from botanical source (bee pollen and honey) and, for later comparisons with similar studies, they were not utilized in this study to collect pollen data, due to the fact that royal jelly is a question of bee secretion. The pollen concentration calculation in royal jelly has no use whatsoever, as it does not come from botanical excretions. In addition, pollen concentration calculations using Lycopodium tracer spores were only carried out in royal jelly in Barth (2005)Barth, O.M. 2005. Botanical resources used by Apis mellifera determined by pollen analysis of royal jelly in Minas Gerais, Brazil. Journal of Apicultural Research 44: 78-81. and Morgado & Barth (2011)Morgado, L. & Barth, O.M. 2011. The detection of pollen in royal jelly of honey bees (Apis mellifera). Journal of ApiProduct and ApiMedical Science 3: 137-139. studies, and even so, without an important discussion about it. Lycopodium tracer spores were not used in royal jelly in the studies conducted by Chauvin & Louveaux (1956)Chauvin, R. & Louveaux, J. 1956, Etude macroscopique et microscopique de la gelée royale. L’apiculteur 1956: 33-43., Ricciardelli D’Albore et al. (1977)Ricciardelli D’Albore, G., Battaglini, M. & D’Ambrosio, M. 1977. Considerazioni sui pollini della gelatina reale. Annali dell’Istituto Sperimentale per la Zoologia Agraria 5: 203-213., Ricciardelli-D’Albore & Battaglini (1978)Ricciardelli-D’Albore, G. & Battaglini, M. 1978. Origine géographique de la gelée royale. Apidologie 9: 1-17., Biondi et al. (2003)Biondi, C., Bedini, G. & Felicioli, A. 2003, Gelatina reale: metodologia proposta per la determinazione dell’origine geografica e della qualità. Apitalia 6: 32-37., Piana et al. (2006)Piana, M.L., Belligoli, P., Persano Oddo, L. & Piperno, S. 2006. Pollen analysis of royal jelly: contribution to Analytical methods and characterization. Apiacta 41: 28-43., Dimou et al. (2007)Dimou, M., Goras, G. & Thrasyvoulou, A. 2007. Pollen analysis as a means to determine the geographical origin of royal jelly. Grana 46: 118-122., Dimou et al. (2013)Dimou, M., Tananaki, C., Goras, G., Karazafiris, E. & Thrasyvoulou, A. 2013. Melissopalynological analysis of royal jelly from Greece. Grana 52: 106-112.. Another reason for not having done concentration calculation and saturation curve is that this is a product manipulated for commercialization, without possibility to control the field production, as observed in other studies, like e.g., in Dimou et al. (2007)Dimou, M., Goras, G. & Thrasyvoulou, A. 2007. Pollen analysis as a means to determine the geographical origin of royal jelly. Grana 46: 118-122., and there is no way of knowing if these samples suffered some type of filtering before being commercialized. Filtering could have artificially reduced the pollen quantity. Finally, in forensic terms, you never add something to a pollen sample and especially not other spores or pollen.

The similarity between the samples was analyzed using a principal component analysis (PCA) to examine the influence of quantitative and qualitative data on the ordination of the pollen types. The matrix included the absolute value of all pollen types counted in each sample. The FITOPAC software Shepherd (1996)Shepherd, G.J. 1996. Fitopac 1: manual do usuário. Campinas: Departamento de Botânica, Universidade Estadual de Campinas. was used for the transformation of the absolute count using the natural logarithm [log (x + 1)] and the ordination was subsequently performed using the covariance matrix in the PC-ORD 5.0 software (Mccune & Mefford 1999Mccune, B. & Mefford, M.J. 1999. PC-ORD. Multivariate Analysis of Ecological Data. GlenedenBeach, Oregon, MjM Software.). The variability between samples was expressed using the first two axes of the PCA.

The MINITAB 15 software (Minitab Inc., State College, PA, 2003) was used to construct a dendrogram of the percent of similarity between the samples.

The samples were evaluated in two ways to identify its origin: 1) among samples, to verify its similarity, including all the pollen types and their absolute value and, 2) with those from other countries, to compare the pollen types found in each sample together with the presence or absence in royal jelly samples from Brazil (Barth 2005Barth, O.M. 2005. Botanical resources used by Apis mellifera determined by pollen analysis of royal jelly in Minas Gerais, Brazil. Journal of Apicultural Research 44: 78-81., Morgado & Barth 2011Morgado, L. & Barth, O.M. 2011. The detection of pollen in royal jelly of honey bees (Apis mellifera). Journal of ApiProduct and ApiMedical Science 3: 137-139.), China (Piana et al. 2006Piana, M.L., Belligoli, P., Persano Oddo, L. & Piperno, S. 2006. Pollen analysis of royal jelly: contribution to Analytical methods and characterization. Apiacta 41: 28-43.), France (Stocker 2005Stocker, A. 2005. Isolation and characterisation of substances from Royal Jelly. Thèse. Université d’Orléans, Université Technique de Munich. Available in https://tel.archives-ouvertes.fr/tel-00008586. (access in 14-III-2018).
https://tel.archives-ouvertes.fr/tel-000...
), Greece (Dimou et al. 2013Dimou, M., Tananaki, C., Goras, G., Karazafiris, E. & Thrasyvoulou, A. 2013. Melissopalynological analysis of royal jelly from Greece. Grana 52: 106-112.) and Italy (Piana et al. 2006Piana, M.L., Belligoli, P., Persano Oddo, L. & Piperno, S. 2006. Pollen analysis of royal jelly: contribution to Analytical methods and characterization. Apiacta 41: 28-43.) as well as Louisiana honey, United States (Lieux 1975Lieux, M.H. 1975. Dominant pollen types recovered from commercial Louisiana honeys. Economic Botany 29: 87-96.-1977Lieux, M.H. 1977. Secondary Pollen Types Characteristic of Louisiana Honeys. Economic Botany 31: 111-119.-1978Lieux, M.H. 1978. Minor honeybee plants of Louisiana indicated by pollen analysis. Economic Botany 32: 418-432.). This allowed investigating if a possible illegal event occurred that could pose serious environmental risks due to the possibility of dispersing this exotic pathogen in Brazil.

The pollen types were grouped in a table showing their probable geographical origins, habitus (form) of the plant, vernacular names in Portuguese and in English, the presence or absence in the samples and a possible indication of the Brazilian extra origin.

The image captures of pollen grains were obtained using an OLYMPUS BX 50 light microscope with a video camera Olympus U-CMAD-2 and the program CellSens Standard® 1.5. for Windows.

Results and Discussion

We identified 78 pollen types representing 48 genera and 38 families of plants, in eight royal jelly samples (table 1). In addition, there were 16 unidentified pollen types. Some samples contained only very few pollen grains as observed also by Dimou et al. (2013)Dimou, M., Tananaki, C., Goras, G., Karazafiris, E. & Thrasyvoulou, A. 2013. Melissopalynological analysis of royal jelly from Greece. Grana 52: 106-112. in Greece.

Table 1
Frequencies of pollen types in the royal jelly samples with Paenibacillus larvae spores purchased in São Paulo State, Brazil, inspected by the S.I.F. Predominant pollen (P), >45% of the total pollen grains counted; Secondary pollen (S) = 16% to 45%. Obs: values are in percentage. The designations after pollen type name indicate the code used in the PCA.

No samples showed pollen spectra compatible with Brazilian biomes or degraded areas by anthropic action of the country (table 2).

Table 2
Geographical origin, life form, vernacular names, economic data of plant usage, and the main occurring environment in Brazil of the plants identified at the genus level by means of the pollen types found in the royal jelly samples with Paenibacillus larvae spores purchased in São Paulo State, Brazil. Note: “Native” means a plant indigenous to a particular region, “Naturalized” means after being introduced into a region, develops without the need to be treated or cultivated under human intervention and “Exotic” means introduced from abroad, but not fully naturalized. Native and naturalized vegetation quoted by Flora do BrasilFlora do Brasil 2020 (em construção). 2018. Available in http://floradobrasil.jbrj.gov.br/ (access in 15-III-2018).
http://floradobrasil.jbrj.gov.br/...
(Brazilian Flora).

Several pollen types found in the samples were from cultivated plants for food, medicinal or ornamental purposes in many parts of the world (Allium, Apium, Brassicaceae, Daucus, Lilium, Taraxacum, Zea mays, among others). Pollen grains from these plants are not good indicators of phytogeographical origin as they can occur in various continents.

On the other hand, the pollen types Acer negundo, Betula, Castanea, Cistus, Cytisus, Hypecoum, Larix, Nyssa, Quercus, Robinia, Sambucus, Saxifraga, Tilia and Ulmus are excellent indicators of origin of samples outside Brazil.

The pollen spectra observed differed from those reported by Barth (2005)Barth, O.M. 2005. Botanical resources used by Apis mellifera determined by pollen analysis of royal jelly in Minas Gerais, Brazil. Journal of Apicultural Research 44: 78-81. and Morgado & Barth (2011)Morgado, L. & Barth, O.M. 2011. The detection of pollen in royal jelly of honey bees (Apis mellifera). Journal of ApiProduct and ApiMedical Science 3: 137-139. for royal jelly samples produced in Brazil, which include a predominance of Cecropia, Asteraceae and Eucalyptus in the city of Viçosa (Minas Gerais State) and Cecropia, Eucalyptus, Melastomataceae and Mimosa pudica/M. scabrella in Pindamonhangaba (São Paulo State), among other pollen grains with lower frequencies. These groups are very distinct from those of the Northern Hemisphere. The pollen spectra of the samples from Pindamonhangaba are similar to those from Vietnam (Piana et al. 2006Piana, M.L., Belligoli, P., Persano Oddo, L. & Piperno, S. 2006. Pollen analysis of royal jelly: contribution to Analytical methods and characterization. Apiacta 41: 28-43.) due to the presence of Mimosa pudica and Eucalyptus, but with different percentages. Another differentiating factor was the presence of other pollen types that only occur in either one of the countries.

The pollen spectra found in the analyzed samples indicate the presence of pollen grains from exotic plants such as thistles (Cirsium/Carduus), oak (Quercus), chestnut (Castanea), rockrose (Cistus), locusts (Robinia), woodbine or ivy (Parthenocissus), tilia (Tilia) and red clover (Trifolium pratense), among others. Several of these pollen grains are reported in European (Louveaux 1970Louveaux, J. 1970. Atlas photographique d’analyse pollinique des miels. Ministere de L’Agriculture, France., Ricciardelli-D’Albore & Intoppa 2000Ricciardelli-D’Albore, G. & Intoppa, F. 2000. Apoidei e Ambiente. In: G. Ricciardelli d’Albore & F. Intoppa (eds.). Fiori e Api. La flora visitata dalle Api e dagli altri Apoidei in Europa. Calderini Edagricole, Bologna, pp. 12-15.), American (Lieux 1975Lieux, M.H. 1975. Dominant pollen types recovered from commercial Louisiana honeys. Economic Botany 29: 87-96.-1977Lieux, M.H. 1977. Secondary Pollen Types Characteristic of Louisiana Honeys. Economic Botany 31: 111-119.-1978Lieux, M.H. 1978. Minor honeybee plants of Louisiana indicated by pollen analysis. Economic Botany 32: 418-432.) or Chinese honey (Song et al. 2012Song, X.Y., Yao, Y.F. & Yang, W.D. 2012. Pollen Analysis of Natural Honeys from the Central Region of Shanxi, North China. PLoS One 7: e49545.), with some regional variations in frequencies.

The pollen variability among the samples comprised 69.68% of the two first axes in the PCA (figure 1). The first axis summed for 53.85% of the variance due to the high values of Castanea, Brassicaceae, Daucus and Taraxacum pollen types which were found in seven samples, as well as Parthenocissus and Veronica, in six and five samples, respectively (table 3).

Figure 1
Principal Component Analysis performed with the pollen type counting variables in eight royal jelly samples with Paenibacillus larvae spores purchased in São Paulo State, Brazil. Codes for pollen types can be found in Table 1.

Table 3
Pearson and Kendall correlation coefficients for pollen grains counting variables of the first and the second axis of PCA ordination in eight royal jelly samples with Paenibacillus larvae spores purchased in São Paulo State, Brazil.

Among the main pollen grains, those from Castanea were found in all analyzed samples and Predominant (P) in samples GR1, GR5, GR8 and GR10 whereas in samples GR2 and GR9 it was Secondary (S). However, in samples GR3 and GR4 it occurred as an Important minor pollen (I) (table 1).

The chestnut is a deciduous tree native to temperate regions of the Northern Hemisphere (North America, Asia and Europe), but it is cultivated in Brazil in some places in São Paulo State, where there are commercial orchards established as long as 20 years ago in Arapeí, Campinas, Campos de Jordão, Cunha, Itupeva, Itapecerica da Serra, Mogi Mirim, Piedade, Pindamonhangaba, Santo Antônio do Pinhal, São Bento do Sapucaí, São José dos Campos and Taubaté (Bueno & Pio 2014Bueno, S.C.S. & Pio, R. 2014. Castanha tipo portuguesa no Brasil. Revista Brasileira de Fruticultura 36: 16-22.). In Minas Gerais State, the commercial orchards are located in the southern region, in Brazópolis, Conceição dos Ouros, Espírito Santo do Dourado, Pouso Alegre, Jacutinga and Gonçalves (Bueno & Pio 2014Bueno, S.C.S. & Pio, R. 2014. Castanha tipo portuguesa no Brasil. Revista Brasileira de Fruticultura 36: 16-22.). However, as several other pollen types observed in all samples did not belong to Brazilian plants, it can be claimed that all samples analyzed were imported.

Other important pollen types for PCA ordination that did not occur frequently in the samples were Acer negundo, Betula, Cistus, Ligustrum, Myosotis, Nyssa, Prunus, Quercus, Salix, Trifolium incarnatum, Zea mays, some Asteraceae and Fabaceae and some unidentified pollen types (table 1). Many of these are indicators of the Northern Hemisphere origin of the samples (table 2).

Some pollen types that had less importance in the PCA ordination of the samples, but that can be good indicators of a Northern Hemisphere origin were Larix, Rumex, Sambucus, Tilia and Ulmus (figure 1).

The pollen types Nyssa and Mimosa pudica are good indicators of phytogeographical origins recovered in the present study. The observations of these pollen types demonstrate an Asian or North American origin as opposed to a European origin (Piana et al. 2006Piana, M.L., Belligoli, P., Persano Oddo, L. & Piperno, S. 2006. Pollen analysis of royal jelly: contribution to Analytical methods and characterization. Apiacta 41: 28-43., Dimou et al. 2013Dimou, M., Tananaki, C., Goras, G., Karazafiris, E. & Thrasyvoulou, A. 2013. Melissopalynological analysis of royal jelly from Greece. Grana 52: 106-112.).

The pollen detected from Nyssa (“tupelo”) may be explained because trees of this genus are highly nectariferous and are valued by beekeepers due to the high quality of honey produced by bees that explore them. Nevertheless, pollen from species of this genus are generally underrepresented in honey samples (Lieux 1978Lieux, M.H. 1978. Minor honeybee plants of Louisiana indicated by pollen analysis. Economic Botany 32: 418-432.). Some of the Nyssa species are native to eastern North America to Central America. Other species also occur naturally in Southeast Asia from China to the southeastern Himalaya Mountains, but they are lacking in Europe. The pollen grains of these two groups can be differentiated through scanning electron microscopy by the presence of tiny elongated sexine elements (rugulae) in the margins of the colpori of the Asian species, while the American ones do not show them (Göschl 2008Göschl, W. 2008. Beiträge zur Pollenmorphologie ausgewählter rezenter und fossiler Vertreter der Davidiaceae und Nyssaceae. Tese, Magister der Naturwissenschaften, Universität Wien, Viena.). In light microscopy, it is not easy to distinguish them. This pollen type was found in samples GR1, GR5, GR9 and GR10 (table 2). Therefore, according to the total pollen spectrum of each sample, this pollen type can be an excellent indicator of the Asian or Northern American phytogeographical origin of royal jelly.

The presence of the Mimosa pudica pollen grains indicates tropical origin. This species is native to Central and South America, but because it is an invasive weed, it now also occurs in Africa, North America, Asia and Australia.

According to Piana et al. (2006)Piana, M.L., Belligoli, P., Persano Oddo, L. & Piperno, S. 2006. Pollen analysis of royal jelly: contribution to Analytical methods and characterization. Apiacta 41: 28-43., Mimosa pudica is one of the pollen types that occur in Asian royal jelly (specifically from Vietnam) but is absent in 28 samples from Italy, as well as in 82 royal jelly samples from Greece analyzed by Dimou et al. (2013)Dimou, M., Tananaki, C., Goras, G., Karazafiris, E. & Thrasyvoulou, A. 2013. Melissopalynological analysis of royal jelly from Greece. Grana 52: 106-112.. In the samples sold in São Paulo, this pollen type was only found in GR8, as an Important minor pollen (I) (table 1). This sample had 97.33% similarity with the group formed by samples GR1, GR5, GR9, and GR10, which are characterized by the presence of Nyssa pollen type and probably originated from Asia or North America (figure 2).

Figure 2
Dendrogram of similarity among the eight royal jelly samples with Paenibacillus larvae spores purchased in São Paulo State, Brazil.

Sample GR4, with Apiaceae, Brassicaceae, Fagopyrum esculentum, Helianthemum, Lotus corniculatus, Quercus, Salix, Taraxacum, Trifolium, Vicia faba and Vitex, was more similar to royal jelly from China (figure 3), based on seven samples analyzed by Piana et al. (2006)Piana, M.L., Belligoli, P., Persano Oddo, L. & Piperno, S. 2006. Pollen analysis of royal jelly: contribution to Analytical methods and characterization. Apiacta 41: 28-43.. Several of these pollen types were also reported in Chinese honey by Song et al. (2012)Song, X.Y., Yao, Y.F. & Yang, W.D. 2012. Pollen Analysis of Natural Honeys from the Central Region of Shanxi, North China. PLoS One 7: e49545..

Figure 3
Dendrogram of similarity among the eight royal jelly samples with Paenibacillus larvae spores purchased in São Paulo State (Brazil) and those ones from Minas Gerais and São Paulo States (Brazil), China, France, Greece and Italy.

Among the frequent pollen types in sample GR4, Quercus and Fagopyrum esculentum occur in Brazil. Quercus is restricted to Paraná State, used for the cultivation of Shiitake mushrooms on oak logs (Bett & Perondi 2011Bett, C.F. & Perondi, M.A. 2011. Análise do mercado de cogumelos comestíveis e medicinais: uma prospecção de alternativa de renda para a agricultura familiar na região sudoeste do Paraná. Synergismus scyentifica 6: 1-8.). Buckwheat (Fagopyrum esculentum) was introduced in the south of the country during the 1920s by the Slavic immigrants (Gorgen 2013Gorgen, A.V. 2013. Produtividade e qualidade da forragem de milheto (Pennisetum glaucum (L.) R.Br) e de trigo mourisco (Fagopyrum esculentum. Moench) cultivado no Cerrado. Monografia, Universidade de Brasília, Brasília.). However, the pollen spectrum of sample GR4 has no similarity with the pollen spectra of Brazilian royal jelly, honey, or bee pollen (Barth 2004Barth, O.M. 2004. Melissopalynology in Brazil: A review of pollen analysis of honeys, propolis and pollen loads of bee. Scientia Agricola 61: 342-350.-2005Barth, O.M. 2005. Botanical resources used by Apis mellifera determined by pollen analysis of royal jelly in Minas Gerais, Brazil. Journal of Apicultural Research 44: 78-81., Morgado & Barth 2011Morgado, L. & Barth, O.M. 2011. The detection of pollen in royal jelly of honey bees (Apis mellifera). Journal of ApiProduct and ApiMedical Science 3: 137-139.). Therefore, the probable phytogeographical origin of this sample is Asia.

According to the PCA analysis, sample GR4, typically Asian, was similar to sample GR3. They were grouped based on the presence of Castanea, Brassicaceae, Daucus, Lotus corniculatus, Trifolium pratense, Poaceae, Prunus, Solanum and Ulmus. These pollen types are common in honey from Europe, Asia and North America (Lieux 1975Lieux, M.H. 1975. Dominant pollen types recovered from commercial Louisiana honeys. Economic Botany 29: 87-96.-1977Lieux, M.H. 1977. Secondary Pollen Types Characteristic of Louisiana Honeys. Economic Botany 31: 111-119.-1978Lieux, M.H. 1978. Minor honeybee plants of Louisiana indicated by pollen analysis. Economic Botany 32: 418-432., Louveaux 1970Louveaux, J. 1970. Atlas photographique d’analyse pollinique des miels. Ministere de L’Agriculture, France., Song et al. 2012Song, X.Y., Yao, Y.F. & Yang, W.D. 2012. Pollen Analysis of Natural Honeys from the Central Region of Shanxi, North China. PLoS One 7: e49545.).

The pollen spectra showing the presence of cultivated plants Brassicaceae, Castanea, Daucus and Trifolium pratense in samples GR3 and GR4 were also reported by Diaz-Losada et al. (1998)Diaz-Losada, E., Ricciardelli-D’Albore, G. & Saa-Otero, M.P. 1998. The possible use of honeybee pollen loads in characterising vegetation. Grana 37: 155-163. in Italian honey. These authors stated that the location of the apiary near a temperate forest is reflected in Italian honey by the presence of Quercus and Ulmus pollen grains or by the abundance of pollen from prairie herbs such as Trifolium pratense and Lamiaceae or also near areas of moist soil due to the presence of pollen of plants adapted to that environment, e.g., Salix.

The royal jelly sample GR2 had a high percentage of pollen of cultivated plants (Brassicaceae and Castanea), similar to many others, but it was the only one with pollen of several species of cultivated plants as celery (Apium, Apiaceae) and Pleurospermum (Apiaceae), two pollen types of Fabaceae (Trifolium and Fabaceae 2), as well as Primulaceae, a family with several ornamental herbaceous plant species widely cultivated around the world. This sample had 88.42% similarity with the group formed by samples with pollen of Nyssa (GR1, GR5, GR9 and GR10) and Mimosa pudica (GR8) (figure 2), supposedly Asian or North American. As pointed out previously, these pollen types do not occur in European royal jelly.

The set with various pollen grains of exotic plants in the samples was similar to the group of European and Chinese royal jelly having 49.54% similarity with those of Brazilian origin (figures 3-12). This percentage was due to the presence of seven pollen types (Amaranthaceae/Chenopodiaceae, Borreria, Brassicaceae, Mimosa pudica/M. scabrella, Poaceae, Senecio and Solanum) reported in the Brazilian samples analyzed by Barth (2005)Barth, O.M. 2005. Botanical resources used by Apis mellifera determined by pollen analysis of royal jelly in Minas Gerais, Brazil. Journal of Apicultural Research 44: 78-81. and by Morgado and Barth (2011)Morgado, L. & Barth, O.M. 2011. The detection of pollen in royal jelly of honey bees (Apis mellifera). Journal of ApiProduct and ApiMedical Science 3: 137-139. as well as in some of the samples analyzed in the present study.

Figure 4
Light microscopy photographs of pollen grains observed in the royal jelly samples. a-d. Gymnospermae. a-b. Cupressaceae. c-d. Pinaceae, Larix. e-l. Angiospermae monocotyledons. e. Amaryllidaceae, Allium. f-g. Iridaceae. h. Liliaceae, Lilium. i. Poaceae, Zea mays. j-k. Unknown reticulate monocotyledon. l. Unknown rugulate monocotyledon. Scale bars: 10 μm.

Figure 5
Light microscopy photographs of pollen grains observed in the royal jelly samples. a-l. Angiospermae eudicotyledons. a-b. Adoxaceae, Sabumcus. c. Amaranthaceae. d-h. Apiaceae. d-e. Apium. f. Daucus. g-h. Pleurospermum. i-l. Aquifoliaceae, Ilex. Scale bars: 10 μm.

Figure 6
Light microscopy photographs of pollen grains observed in the royal jelly samples. a-l. Angiospermae eudicotyledons, Asteraceae. a. Ambrosia. b-c. Artemisia. d. Cirsium/Carduus. e-f. Senecio. g-h. Taraxacum. i-j. Asteraceae l. k-l. Asteraceae 3. Scale bars: 10 μm.

Figure 7
Light microscopy photographs of pollen grains observed in the royal jelly samples. a.l. Angiospermae eudicotyledons. a. Asteraceae 4. b. Asteraceae 5. c. Betulaceae, Betula. d-e. Brassicaceae. f-g. Boraginaceae, Myosotis. h-i. Caryophyllaceae. h. Caryophyllaceae 1. i. Melandrium. j-l Cistaceae, Cistus. Scale bars: 10 μm.

Figure 8
Light microscopy photographs of pollen grains observed in the royal jelly samples. a-l. Angiospermae eudicotyledons. a-b. Cornaceae, Nyssa. c-f. Euphorbiaceae. g-l. Fabaceae. g-h. Cassia. i-j. Lotus corniculatus. k. Mimosa pudica. l. Robinia. Scale bars: 10 μm.

Figure 9
Light microscopy photographs of pollen grains observed in the royal jelly. a-l. Angiospermae eudicotyledons. a-h. Fabaceae. a-b. Robinia. c. Trifolium incarnatum. d-e. Trifolium repens. f. Vicia faba. g. Fabaceae 1. h. Fabaceae 4. i-l. Fagaceae. i-j. Castanea. k-l. Quercus. Scale bars: 10 μm.

Figure 10
Light microscopy photographs of pollen grains observed in the royal jelly samples. a-l. Angiospermae eudicotyledons. a-b. Gentianaceae. c. Lamiaceae. d-e. Malvaceae, Tilia. f-i. Meliaceae. f. Meliaceae 1. g-h. Meliaceae 2. i. Meliaceae 3. j-k. Oleaceae, Ligustrum. l. Polygonaceae, Rumex. Scale bars: 10 μm.

Figure 11
Light microscopy photographs of pollen grains observed in the royal jelly samples. a-l. Angiospermae eudicotyledons. a-c. Plantaginaceae, Veronica. d-e. Primulaceae, f-i. Ranunculaceae. f-g. Caltha. h-i. Thalictrum. j-k. Rosaceae, Prunus. l. Salicaceae, Salix. Scale bars: 10 μm.

Figure 12
Light microscopy photographs of pollen grains observed in the royal jelly samples. a-l. Angiospermae eudicotyledons. a. Salicaceae, Salix. b-c. Sapindaceae, Acer negundo. d-f. Saxifragaceae, Saxifraga. g-h. Solanaceae. i. Ulmaceae, Ulmus. j-l. Vitaceae, Parthenocissus. Scale bars: 10 μm.

Palynological evidence indicates that royal jelly samples contaminated with spores of P. larvae are sold in São Paulo State as Brazilian products, however, they were produced in the Northern Hemisphere. The fact that royal jelly samples from Europe analyzed by other authors have been found similar to those from China, due to the presence of some pollen types, suggests that palynology alone cannot accurately identify the continent or country of origin. No scientific specialty (plant anatomy, palynology, physicochemical) alone can solve problems of the origin of bee products and illegal imports of bee products. Thus, only field observations of bees’ visits to flowers cannot define the botanical origin of the floral resources used by bees. Beekeepers’ empirical observations should be followed up by physicochemical, anatomical and palynological analyses of bee products, as these analyses broaden the knowledge about bee visits to flowers. In order to be sure of the geographical origin of these contaminated royal jelly batches, public inspection agents should audit in the warehouses that packed the royal jelly about the origin of the contaminated product that is being marketed in São Paulo.

The Brazilian Ministry of Agriculture had no records of imported royal jelly from Europe, North America or Asia related to the period of the corresponding lots. Thus, the contaminated lots were probably illegally imported, threatening national beekeepers. As royal jelly production in Brazil is no longer economically viable due to the illicit entry of royal jelly over the past decade from China at low prices, we suggest that the analyzed products are original without the mixture of royal jelly produced locally in Brazil. This is a matter of concern because the dispersion of P. larvae spores from imported products without sanitary inspection is associated with the following contamination routes: using of contaminated royal jelly to feed queen larvae; manipulating contaminated products and/or utensils used to handle healthy colonies; using contaminated wax as comb substrate; access of bees to remains of contaminated imported products discarded near apiaries; cross-contamination in warehouses (e.g., pollen dryers, where pollen grains of different origins are dehydrated) and supply of contaminated food supplements to healthy bees during the winter (especially bee pollen).

The possibility of P. larvae dispersion and contamination of healthy colonies is aggravated by traditional handling procedures such as transferring wax frames among colonies, using the same utensils in multiple bee hives and transferring colonies among apiaries, as well the natural trophallaxis among bees.

We stress the urgency of regulatory agencies taking appropriate measures at warehouses to verify the origin of the lots of royal jelly marketed and thus detect and avoid the illicit trade, as well as to implement risk analysis to identify critical points of the chain for traceability.

Conclusions

No royal jelly sample contaminated with Paenibacillus larvae had a pollen spectrum compatible with Brazilian vegetation, showing that the products analyzed were imported. The pollen types identified in the samples indicated probable phytogeographical origin from the Northern Hemisphere, either from Europe, Asia or North America.

By carrying out a palynological analysis, it was observed that the pollen grains in the royal jelly samples are from species occurring in temperate climates, such as boreal forests with conifers and milder climates, for example in the Mediterranean. Pollen grains from forage grasses, as well various crops were observed in the royal jelly samples showing similarity with the samples produced by Apis mellifera bees in most parts of the world. Therefore, these pollen types are not appropriate for the characterization of the phytogeographical origin of royal jelly.

The presence of P. larvae spores in bee products from Brazilian apiaries is an unexpected finding because the country is officially considered free from the disease. However, a previous study (molecular analysis) done for pathogen detection in the same samples showed that in São Paulo State royal jelly has spores of this bacterium. The lack of effective control in this commercialization, as proven in this study, can ruin Brazilian beekeeping if this pathogen is spread throughout the country and applied palynology in forensic science is helping in this battle.

Overall, our findings show that palynology is a powerful tool in identifying the phytogeographic origins of royal jelly from the Northern Hemisphere. Although the current legislation does not require bee products to be examined palynologically to verify phytogeographic origins, this analysis can help to control pathogens from spreading. In addition, we show the fragility of actions regarding the control of imported products and thus the urgent need for more effective rules and actions by sanitary authorities.

Acknowledgements

This work was supported by the São Paulo State Research Foundation (FAPESP) under Grant (number 2012/18802-3). CFPL is researcher fellow of the National Council for Scientific and Technological Development (CNPq) under Grant (number 302766/2016-2). MFM is a researcher fellow of the National Council for Scientific and Technological Development (CNPq). JES is a research fellow of the CNPq and Minas Gerais State Research Foundation (FAPEMIG). CFPL also thanks FAPESP for providing financial support to her participation in the Mediterranean Palynology 2015 Symposium (Process Number 2015/145997). Jane Godwin Coury translated the English manuscript.

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Publication Dates

  • Publication in this collection
    Jul-Aug 2018

History

  • Received
    27 Apr 2018
  • Accepted
    11 July 2018
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