Bombus brasiliensis Lepeletier (Hymenoptera, Apidae) infected with Nosema ceranae (Microsporidia)

ABSTRACT

Heavy infections caused by a microsporidium were detected in midgut epithelium cells of two adult workers of the bumble bee Bombus brasiliensis Lepeletier collected near Puerto Iguazú, Misiones province, Argentina. Microsporidium rRNA (16S small subunit) was amplified by 218MITOC primers and produced amplicons indicating presence of Nosema ceranae Fries et al., a virulent pathogen of more than 20 bee species, possibly involved in Apis mellifera L. Colony Collapse Disorder. Campaigns in search of B. brasiliensis between 2008 and 2015 have revealed a possible narrower range in the southeastern area of its known distribution. Effects of N. ceranae infections could be modulating their populations and should not be overlooked. In addition, the wide host range of this microsporidium makes it a potential threat to several endemic bees such as stingless (Meliponini) and orchid bees (Euglossini).

Keywords:
Argentina; Brazil; Bumble bee; Colony Collapse Disorder

When compared to other regions of the world, southern South America seems to depict low bumble bee diversity (Williams, 1998). Only ten out of the ca. 250 species of Bombus described worldwide have been reported to inhabit Argentina. Two of them, Bombus ruderatus (Fabricius, 1775) and B. terrestris (L., 1758), are invasive species of relatively recent entry into the southwest of the country from Chile, while the remaining eight are native ( Abrahamovich et al., 2007 and Schmid-Hempel et al., 2014).

According to the last available surveys on their geographic distribution in Argentina (Moure and Sakagami, 1962, Abrahamovich and Díaz, 2001, Abrahamovich et al., 2004 and Abrahamovich et al., 2007), B. pauloensis Friese, 1913 (=B. atratus Franklin, 1913) , B. bellicosus Smith, 1879, B. morio (Swederus, 1787), and B. opifex Smith, 1879 are known to exhibit wide ranges. Bombus tucumanus Vachal, 1904, B. baeri Vachal, 1904, and B. dahlbomii Guérin, 1835 appear to show more limited ranges, while B. brasiliensis Lepeletier, 1836 may possibly occur only in Misiones province at the northeastern tip of the country. Although B. brasiliensis, an assiduous visitor of bromeliad flowers (Bromeliaceae) like Aechmea spp. ( Kaehler et al., 2005 and Schmid et al., 2011) appears to be widespread in Brazil ( Abrahamovich et al., 2004 and Santos Júnior et al., 2015), surveys carried out by our group since January 2008 suggest that its distribution in Argentina may be nowadays considerably reduced. This communication reports the detection of the microsporidium Nosema ceranae infecting B. brasiliensis and argues on a possible effect on the distribution of this bee species on the southeastern part of its range.

After campaigns in search of B. brasiliensis since 2008 done by authors and other team members surveying 32 localities in the provinces of Formosa (Bañado La Estrella, Colonia Perin, El Colorado, Gran Guardia, Ibarreta, Ingeniero Juárez, Laguna Yema, Las Lomitas, Palo Santo, Pirané, Posta Cambio Zalazar, Pozo del Tigre), Chaco (38 km North of Resistencia, Colonia Elisa, Juan José Castelli, Resistencia, Presidencia Roque Sáenz Peña), Corrientes (Colonia Carlos Pellegrini, Corrientes, Estero Santa Lucía, Laguna Iberá, Santo Tomé), and Misiones (Aristóbulo del Valle, Cuña Pirú, El Alcázar, 30 km East of María Magdalena, Montecarlo, Leandro N. Alem, Posadas, Urugua-í, Wanda) (Fig. 1A, Table 1) with no positive results, only six adult workers were collected in February 2015 in the surroundings of Puerto Iguazú, Misiones, northeastern Argentina (Fig. 1A). They were captured while foraging using entomological nets, conserved frozen (-32° C), and identified based on information provided by Moure and Sakagami (1962), Abrahamovich et al. (2005), and Santos Júnior et al. (2015).

Fig. 1
(A) The 32 surveyed localities in northeastern Argentina. Formosa province [Fo]: (1) Ingeniero Juárez; (2) Laguna Yema; (3) Las Lomitas; (4) Bañado La Estrella; (5) Posta Cambio Zalazar; (6) Pozo del Tigre; (7) Colonia Perin; (8) Ibarreta; (9) Palo Santo; (10) Pirané; (11) Gran Guardia; (12) El Colorado. Chaco province [Ch]: (13) J. J. Castelli; (14) Presidencia Roque Saenz Peña; (15) Colonia Elisa; (16) 38 km North of Resistencia; (17) Resistencia. Corrientes province [Co]: (18) Corrientes; (19) Estero Santa Lucía; (20) Colonia Carlos Pellegrini; (21) Laguna Iberá; (22) Santo Tomé. Misiones province [Mi]: (23) Posadas; (24) Leandro N. Alem; (25) Cuña Pirú; (26) Aristóbulo del Valle; (27) El alcázar; (28) Montecarlo; (29) 30 km East of María Magdalena; (30) Wanda; (31) Urugua-í. (★) indicates Puerto Iguazú, the only locality where six Bombus brasiliensis workers were found. (B) Habitat where B. brasiliensis was found. (C) Two distended midgut cells of B. brasiliensis with spores of Nosema ceranae inside.

Table 1
Date and location of the 32 surveyed localities in northeastern Argentina looking for Bombus brasiliensis. Bold indicates the only campaign with findings (see text for details).

Province	Locality	GPS location	Date
Chaco	38 km North of Resistencia	27°05'10" S; 58°57'24" W	Feb 2015
	Colonia Elisa	26°51'57" S; 59°31'51" W	Jan 2008
	J. J. Castelli	25°54'02" S; 60°32'06" W	Feb 2012 Feb 2015
	Presidencia Roque Saenz Peña	26°47'04" S; 60°28'17" W	Jan 2008 Feb 2012 Feb 2015
	Resistencia	27°23'05" S; 58°59'22" W	Feb 2015
Corrientes	Colonia Carlos Pellegrini	28°32'14" S; 57°11'04" W	Jan 2008 Oct 2009
	Corrientes	27°27'38" S; 58°47'35" W	Jan 2008 Feb 2012 Feb 2015
	Estero Santa Lucía	28°01'15" S; 58°01'35" W	Jan 2008
	Laguna Iberá	28°30'01" S; 57°04'50" W	Oct 2009
	Santo Tomé	28°32'41" S; 56°01'45" W	Feb 2015
Formosa	Bañado La Estrella	24°30'32" S; 60°25'36" W	Feb 2015
	Colonia Perin	25°36'08" S; 60°04'15" W	Feb 2015
	El Colorado	26°19'57" S; 59°21'37" W	Feb 2015
	Gran Guardia	25°50'19" S; 58°48'24" W	Feb 2012
	Ibarreta	25°11'49" S; 58°48'24" W	Feb 2012 Feb 2015
	Ingeniero Juárez	23°55'230" S; 61°47'58" W	Feb 2015
	Laguna Yema	24°11'10" S; 61°18'48" W	Feb 2015
	Las Lomitas	24°43'58" S; 60°33'22" W	Feb 2012 Feb 2015
	Palo Santo	25°33'57" S; 59°16'52" W	Feb 2012 Feb 2015
	Pirané	25°40'24" S; 59°05'50" W	Feb 2012 Feb 2015
	Posta Cambio Zalazar	24°12'47" S; 60°11'46" W	Feb 2015
	Pozo del Tigre	24°53'55" S; 60°18'25" W	Feb 2012 Feb 2015
Misiones	30 km East of María Magdalena	26°11'26" S; 54°17'17" W	Feb 2015
	Aristóbulo del Valle	27°07'02" S; 54°52'45" W	Jan 2008
	Cuña Pirú	27°05'17" S; 54°57'09" W	Jan 2008
	El alcázar	26°43'43" S; 54°47'55" W	Jan 2008
	Leandro N. Alem	27°35'17" S; 55°15'00" W	Jan 2008
	Montecarlo	26°33'42" S; 54°43'29" W	Sept 2009 Feb 2015
	Posadas	27°24'04" S; 55°55'23" W	Jan 2008 Feb 2015
	Puerto Iguazú	25°38'52" S; 54°32'40" W	Jan 2008 Feb 2015
	Urugua-í	25°52'24" S; 54°33'31" W	Jan 2008 Feb 2015
	Wanda	25°57'05" S; 54°34'32" W	Jan 2008 Feb 2015

Examination of each individual was performed following dissection techniques under stereoscopic microscopy (×10, ×40) (Lacey and Solter, 2012). Briefly, small portions of different tissues and organs were extracted in order to prepare fresh smears with one-quarter-strength Ringer's solution (Poinar and Thomas, 1984) for detection of microsporidia and protists (Lange and Lord, 2012 and Solter et al., 2012). Observations were done using phase-contrast microscopy (×400, ×1000). Each infected individual was then homogenized in 2 mL of double distilled water and infection intensity (spore load) was quantified using an Improved Neubauer hemocytometer (Undeen and Vávra, 1997). Spore suspensions were obtained by repeated filtration and centrifugation (15 min; 7500 × g) ( Lange and Henry, 1996). Double distilled water was replaced by absolute ethanol in spore suspensions and stored at -32 °C until genetic analysis were performed. Microsporidium rRNA (16S small subunit) was amplified by real time PCR according to Medici et al. (2012) with specific primers for Nosema apis Zander, 1907 (321APIS) and N. ceranae (218MITOC). Amplicons were separated on ethidium bromide-stained 1% agarose gel. Genetic material was purified with an ExoSap-IT kit (Amersham, Biosciences) and sequenced in an automatic MegaBACE Sequence Analyzer (Amersham, Biosciences). Sequences were aligned using SMS software ( Stothard, 2000) and submitted to Genbank.

Microsporidian infections were detected in two individuals of B. brasiliensis collected while foraging on Solanum sp. (Solanaceae) in a farmland 5 km southeast of Puerto Iguazú (25°38'52" S; 54°32'40" W) (Fig. 1B). Diseased individuals did not show obvious external signs of infection. The microsporidium was found infecting cells of the midgut epithelium, many of which were distended due to the heavy presence of spores (Fig. 1C). Infection intensity for each bumble bee was 5.5 × 10⁷ and 4.4 × 10⁷ spores/insect. Samples of spores from both infections were amplified by 218MITOC primers and produced amplicons indicating presence of N. ceranae. One of the sequences (220 bp) was deposited on GeneBank under accession number KX024757.

Nosema ceranae was originally described from the Asian honey bee Apis cerana Fabricius, 1793 ( Fries et al., 1996). Ten years after its description, it was also detected in the European honey bee A. mellifera L., 1758 in Spain ( Higes et al., 2006). Teixeira et al. (2013) evidenced that N. ceranae has been present in Brazil for at least 36 years infecting Africanized honey bees. Plischuk et al. (2009) reported for the first time the presence of this microsporidium in bumble bees infecting three native South American species of genus Bombus [B. pauloensis (named as B. atratus), B. bellicosus, B. morio].

Transmission of N. ceranae is mainly horizontal ( Higes et al., 2008). This microsporidium completes its lifecycle inside midgut epithelial cells. Spores leave the host with the feces and may remain viable in the environment until they enter a new individual per os. During the last decade, numerous studies worldwide have demonstrated high virulence of N. ceranae against A. mellifera ( Paxton et al., 2007 and Higes et al., 2007), suggesting a role in the honey bees Colony Collapse Disorder (CCD) (Higes et al., 2008). Effects of N. ceranae in honey bees are relatively well known and include lesions in the midgut epithelium causing metabolic stress and suppressing immune response ( Antúnez et al., 2009 and Mayack and Naug, 2009). Higes et al. (2007) have reported 100% bee mortality 8 days post inoculation under experimental conditions. On the contrary, pathological effects of N. ceranae in bumble bees are not completely clear. Experimental infections on B. terrestris have shown that this pathogen would be highly virulent, colonizing the midgut epithelium and reducing host survival by 48% within one week after exposure ( Graystock et al., 2013).

Because these are the first detections of N. ceranae in B. brasiliensis, effects on the host are yet unknown. However based on the observation that infections in both individuals were advanced and heavy, a significant virulence possibly similar to that described in other hosts would not be unexpected.

An important subject is the possible spread of N. ceranae to other species. Since the vectoring of pathogens by the sharing use of flowers seems to be a relatively common process, the transferring of infective spores between bees and other pollinators appears highly likely ( Graystock et al., 2013 and Graystock et al., 2015), and B. brasiliensis [possibly along with A. mellifera ( Teixeira et al., 2013)] could act as source of N. ceranae to other sympatric species. Recent studies have demonstrated that N. ceranae is capable to infect not only Bombus and Apis species but also solitary bees belonging to genus Andrena (Andrenidae), Osmia, and Heriades (Megachilidae) in Belgium ( Ravoet et al., 2014). Numerous endemic bees inhabit the Neotropical region, as several stingless bees (Meliponini) (Freitas et al., 2009) and orchid bees (Euglossini) (Nemésio, 2009) that could be potential hosts. If these bees are susceptible, the effects of N. ceranae might be enhanced as seen in other, several new host-parasite associations ( Goulson and Hughes, 2015).

Although data about the presence of B. brasiliensis is fragmented, the extension of its geographic distribution appears to be from the southern portions of the Araguaia-Tocantins basin in Mato Grosso, Brazil to northern Argentina and Uruguay in the South. The western limit would be along the Paraná River basin (Mato Grosso do Sul and southern Goiás), whereas to the East it would reach the Atlantic coast ( Santos Júnior et al., 2015).

However, recent studies have shown that the eastern range would actually be narrower than previously thought since that area is inhabited by B. bahiensisSantos Júnior et al., 2015, a recently described sister species ( Santos Júnior et al., 2015). The southwestern range into Argentina also seems to have historically diminished from Chaco, Formosa and Misiones provinces ( Holmberg, 1903, Moure and Sakagami, 1962, Abrahamovich and Díaz, 2001 and Abrahamovich et al., 2004) to only the latter (Abrahamovich et al., 2007). Absence of detections of B. brasiliensis after several campaigns since 2008 at more than 30 localities ( Fig. 1A, Table 1) within the original range may suggests a more restricted presence nowadays, apparently limited to a small area at the northeastern tip of the country (Fig. 1A). Bombus brasiliensis was also present in Uruguay during the last century ( Moure and Sakagami, 1962) but it has not been detected in that country since at least 2005, suggesting that the southernmost distribution could also have suffered a retraction (Santos et al., 2013). In Uruguay, N. ceranae is highly prevalent in B. pauloensis and B. bellicosus ( Arbulo et al., 2015).

Although evidencing a cause-effect link between occurrence of N. ceranae and the apparent range retraction of B. brasiliensis constitute an elusive goal, we feel it is important to report on the infections we have found. Nosema ceranae may be at least one of the drivers modulating the populations of this host.

Acknowledgements

Authors are grateful to C. Grundler, A. Panizza and A. Martins. Special thanks to C. Bardi, M. Pocco, S. Pelizza and C. Scattolini for their help in field work, and to S. Medici for technical assistance. This study was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Comisión de Investigaciones Científicas (CICPBA), and Agencia Nacional de Promoción Científica y Tecnológica (PICT 2012-0851; PICT 2012-0199).

References

• Abrahamovich, A.H., Díaz, N.B., 2001. Distribución geográfica de las especies del género Bombus Latreille (Hymenoptera, Apidae) en Argentina. Rev. Bras. Ento- mol. 45, 23-36.
• Abrahamovich, A.H., Díaz, N.B., Morrone, J.J., 2004. Distributional patterns of the neotropical and Andean species of the genus Bombus (Hymenoptera, Apidae). Acta Zool. Mex. 20, 99-117.
• Abrahamovich, A.H., Díaz, N.B., Lucía, M., 2005. The species of the genus Bombus Latreille in Argentina (Hymenoptera, Apidae): taxonomic study and keys for their identification. Neotrop. Entomol. 34, 235-250.
• Abrahamovich, A.H., Díaz, N.B., Lucía, M., 2007. Identificación de las abejas sociales del género Bombus (Hymenoptera, Apidae) presentes en la Argentina: clave pic- tórica, diagnosis, distribución geográfica y asociaciones florales. Rev. Fac. Agron. La Plata 106, 165-176.
• Antúnez, K., Martín-Hernández, R., Prieto, L., Meana, A., Zunino, P., Higes, M., 2009. Immune suppression in the honey bee (Apis mellifera) follow- ing infection by Nosema ceranae (Microsporidia). Environ. Microbiol. 11, 2284-2290.
• Arbulo, N., Antúnez, K., Salvarrey, S., Santos, E., Branchiccela, B., Martín-Hernández, R., Higes, M., Invernizzi, C., 2015. High prevalence and infection levels of Nosema ceranae in bumblebees Bombus atratus and Bombus bellicosus from Uruguay. J. Invertebr. Pathol. 130, 165-168.
• Freitas, B.M., Imperatriz-Fonseca, V.L., Medina, L.M., Kleinert, A.M.P., Galetto, L., Nates-Parra, G., Quezada-Euán, J.J.G., 2009. Diversity, threats and conservation of native bees in the Neotropics. Apidologie 40, 332-346.
• Fries, I., Feng, F., da Silva, A., Slemenda, S.B., Pieniazek, N.J., 1996. Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). Eur. J. Protistol. 32, 356-365.
• Goulson, D., Hughes, W.O., 2015. Mitigating the anthropogenic spread of bee para- sites to protect wild pollinators. Biol. Conserv. 191, 10-19.
• Graystock, P., Yates, K., Darvill, B., Goulson, D., Hughes, W.O.H., 2013. Emerging dan- gers: deadly effects of an emergent parasite in a new pollinator host. J. Invertebr. Pathol. 114, 114-119.
• Graystock, P., Goulson, D., Hughes, W.O.H., 2015. Parasites in bloom: flowers aid dis- persal and transmission of pollinator parasites within and between bee species. Proc. R. Soc. B 282, 20151371.
• Higes, M., Martín, R., Meana, A., 2006. Nosema ceranae, a new microsporidian par- asite in honeybees in Europe. J. Invertebr. Pathol. 92, 81-83.
• Higes, M., García-Palencia, P., Martín-Hernández, R., Meana, A., 2007. Experimental infection of Apis mellifera honeybees with Nosema ceranae (Microsporidia). J. Invertebr. Pathol. 94, 211-217.
• Higes, M., Martín-Hernández, R., Botías, C., Garrido Bailón, E., González-Porto, A.V., Barrios, L., del Nozal, M.J., Bernal, J.L., Jiménez, J.J., García Palencia, P., Meana, A., 2008. How natural infection by Nosema ceranae causes honeybee colony collapse. Environ. Microbiol. 10, 2659-2669.
• Holmberg, E.L., 1903. Delectus Hymenopterologicus Argentinus. Hymenopterorum Argentinorum et quorumdam exoticorum observationes synonimicas, adden- das, novorumque generum specierumque descriptiones contines. An. Mus. Nac. B. Aires 3, 377-468.
• Kaehler, M., Varassin, I.G., Goldenberg, R., 2005. Pollination of a bromeliad commu- nity in the high montane Atlantic rain forest in Paraná state, Brazil. Rev. Brasil. Bot. 28, 219-228.
• Lacey, L.A., Solter, L.F., 2012. Initial handling and diagnosis of diseased invertebrates. In: Manual of Techniques in Invertebrate Pathology, 2nd ed. Elsevier Ltd., Lon- don, pp. 1-14.
• Lange, C.E., Henry, J.E., 1996. Métodos de estudio y producción de protistas ento- mopatógenos. In: Lecuona, R. (Ed.), Microorganismos patógenos empleados en el control microbiano de plagas. M. Mas., Bs. As, pp. 169-176.
• Lange, C.E., Lord, J.C., 2012. Protistan entomopathogens. In: Vega, F., Kaya, H. (Eds.), Insect Pathology and Microbial Control. Academic Press, San Diego, pp. 367-394.
• Mayack, C., Naug, D., 2009. Energetic stress in the honeybee Apis mellifera from Nosema ceranae infection. J. Invertebr. Pathol. 100 (3), 185-188.
• Medici, S.K., Sarlo, E.G., Porrini, M.P., Braunstein, M., Eguaras, M.J., 2012. Genetic variation and widespread dispersal of Nosema ceranae in Apis mellifera apiaries from Argentina. Parasitol. Res. 110 (2), 859-864.
• Moure, J.S., Melo, G.A.R., 2012. Bombini Latreille, 1802. In: Moure, J.S., Urban, D., Melo, G.A.R. (Eds.), Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropi-cal Region. , Available from: http://www.moure.cria.org.br/catalogue (accessed 05.04.16).
  » http://www.moure.cria.org.br/catalogue
• Moure, J.S., Sakagami, S.F., 1962. As mamangabas sociais do Brasil (Bombus Latreille) (Hymenoptera, Apoidea). Studia Entomol. 5, 65-194.
• Nemésio, A., 2009. Orchid bees (Hymenoptera, Apidae) of the Brazilian Atlantic Forest. Zootaxa 2041, 1-242.
• Paxton, R.J., Klee, J., Korpela, S., Fries, I., 2007. Nosema ceranae has infected Apis mellifera in Europe since at least 1998 and may be more virulent than Nosema apis. Apidologie 38, 558-565.
• Plischuk, S., Martín-Hernández, R., Prieto, L., Lucía, M., Botías, C., Meana, A., Abrahamovich, A.H., Lange, C.E., Higes, M., 2009. South American native bum- blebees (Hymenoptera, Apidae) infected by Nosema ceranae (Microsporidia), an emerging pathogen of honeybees (Apis mellifera). Environ. Microbiol. Rep. 1, 131-135.
• Poinar, G.O., Thomas, G.M., 1984. Laboratory Guide to Insect Pathogens and Parasites. Plenum Press, New York.
• Ravoet, J., De Smet, L., Meeus, I., Smagghe, G., Wenseleers, T., de Graaf, D.C., 2014. Widespread occurrence of honey bee pathogens in solitary bees. J. Invertebr. Pathol. 122, 55-58.
• Santos, E., Arbulo, N., Salvarrey, S., Invernizzi, C., 2013. First study of distribution of species of the genus Bombus (Hymenoptera, Apidae) in Uruguay. In: Proceed. 43rd Apimondia Cong. Kyiv, Ukranie, p. 360.
• Santos Júnior, E.S., Santos, F.R., Silveira, F.A., 2015. Hitting an unintended target: phylogeography of Bombus brasiliensis Lepeletier, 1836 and the first new Brazil- ian bumblebee species in a century (Hymenoptera: Apidae). PLOS ONE 10 (5), e0125847.
• Schmid, S., Schmid, V.S., Zillikens, A., Harter-Marques, B., Steiner, J., 2011. Bimodal pollination system of the bromeliad Aechmea nudicaulis involving humming- birds and bees. Plant Biol. 13 (Suppl. 1), 41-50.
• Schmid-Hempel, R., Eckhardt, M., Goulson, D., Heinzmann, D., Lange, C., Plischuk, S., Ruz Escudero, L., Salathé, R., Scriven, J., Schmid-Hempel, P., 2014. The invasion of southern South America by imported bumblebees and associated parasites. J. Anim. Ecol. 83, 823-837.
• Solter, L.F., Becnel, J.J., Oi, D.H., 2012. Microsporidian Entomopathogens. In: Vega, F., Kaya, H. (Eds.), Insect Pathology and Microbial Control. Academic Press, San Diego , pp. 221-264.
• Stothard, P., 2000. The sequence manipulation suite: JavaScript programs for ana- lyzing and formatting protein and DNA sequences. Version 2. Biotechniques 28, 1102-1104.
• Teixeira, E.W., dos Santos, L.G., Sattler, A., Message, D., Alves, M.L.T.M.F., Martins, M.F., Grassi-Sella, M.L., Francoy, T.M., 2013. Nosema ceranae has been present in Brazil for more than three decades infecting Africanized honey bees. J. Invertebr. Pathol. 114, 250-254.
• Undeen, H.H., Vávra, J., 1997. Research methods for entomopathogenic Protozoa. In: Lacey, L. (Ed.), Manual of Techniques in Insect Pathology. Academic Press, New York, pp. 117-151.
• Williams, P.H., 1998. An annotated checklist of bumble bees with an analysis of patterns of description (Hymenoptera: Apidae, Bombini). Bull. Nat. Hist. Mus. Lond. 67, 79-152.

• 1
  Although the name B. atratus is widely adopted, the valid name seems to be B. pauloensis. See Moure and Melo (2012) for detailed information.

Publication Dates

• Publication in this collection
  Oct-Dec 2016

History

• Received
  06 Jan 2016
• Accepted
  03 June 2016