Viruses in Brazilian stingless bees

Abstract Stingless bees are important pollinators in tropical and subtropical areas. The breeding of native stingless bee species from the Meliponini tribe to obtain honey, pollen, and geopropolis (meliponiculture) is a promising activity in Brazil with economic, social, and environmental impacts. Stingless bees can share food sources with Apis mellifera and other insects that can be vectors of pathogens. Here, we investigated seven stingless bee species from Brazil (Melipona quadrifasciata anthidioides, Melipona scutellaris, Nannotrigona testaceicornis, Partamona helleri, Scaptotrigona xanthotricha, Tetragonisca angustula, and Trigona spinipes) screening for seven of the most common pathogenic viruses (ABPV, IAPV, BQCV, CBPV, SBV, KBV, and DWV) in A. mellifera in summer and winter seasons. All seven stingless bee species studied were positive for ABPV and IAPV (only in M. quadrifasciata anthidioides and T. spinipes), while ABPV was found in samples from summer and winter, IAPV was only found in winter. This is the first study detecting viruses in seven native stingless bee species in Brazil and because of the novelty of this area in Brazil, it is necessary to monitor native stingless bee colonies to investigate the impact of those viruses at a colony and individual level to avoid losses and impact to the native bees.


Introduction
Social stingless bees are important pollinators (Garibaldi et al., 2013) and can be found in tropical and subtropical areas.Eighty percent of the total species are concentrated in Central and Southern America (Camargo et al., 2023), and 60% in Brazil (Pedro, 2014).
Meliponiculture or the practice of keeping stingless bees is well established in Brazil and practiced across the whole country, where the most important product is honey with the most common species maintained: Tetragonisca angustula, Melipona quadrifasciata anthidioides, M. scutellaris, M. subnitida, and M. fasciculata (Jaff� e et al., 2015).However, the main bee used for beekeeping in Brazil is the exotic species Apis mellifera, which is host to several viral pathogens.Due to its wide distribution Apis mellifera becomes a natural reservoir that can transmit viruses to other species of bees (Goulson, 2003) via interactions between them and the environment by sharing food sources, which can contribute to the horizontal transmission of pathogens from A. mellifera to stingless bees (Graystock et al., 2015;Mcart et al., 2014;Purkiss & Lach, 2019).
The viruses that can infect stingless bees are not yet well known (De Paula et al., 2021), and in Brazil, this area is still limited, however, beekeepers are recognizing the impact that diseases can have on their colonies (Freitas et al., 2022), and the need to identify the origin of this possible diseases.In this context, we aimed to evaluate the presence of viral pathogens in Brazilian native stingless bee species commonly used in meliponiculture.
The samples were preserved in alcohol 70% and kept in an ultrafreezer at −80 � C until sample processing.A pool of 30 stingless bees (per colony) was crushed (thorax and abdomen) to a fine powder using liquid nitrogen, transferred to cryotubes, and kept in ultrafreezer at −80 � C prior to analysis.

RNA extraction and RT-PCR
RNA extraction was performed using RNAzol RT (Invitrogen, Carlsbad, CA, US) following the manufacturer's protocol, with 1000 lL of RNAzol added to 100 mg of crushed bees in 1.5 mL microtubes for cell lysis.RNA was precipitated by adding 400 lL of absolute isopropyl alcohol and leaving the sample to rest for 10 min, following 4 washes with 400 lL of ethyl alcohol 75% prior to precipitation.RNA was eluted in 50 ll of ultrapure water and quantified using Biophotometer D30 (Eppendorf, Hamburg, DE), with absorbance values at 260/280 mm ranging from 1.7 to 2.0.RNA purification was performed using DNA-free Kit (Invitrogen, Carlsbad, CA, US) according to the manufacturer's protocol.First-strand cDNA synthesis and PCR were carried out using SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, CA, US) according to the configurations (Table S1) of each specific primer (Table S2), using 4 lL of RNA extracted (corresponding to 30 ng RNA, approximately).The reaction was performed in Veriti 96-Well Thermal-Cycler (Applied Biosystems, MA, US).Ultrapure water was used as negative control and for positive controls were used positive RNA results from Apis mellifera (Peixoto et al., 2021).RT-PCR product was performed by 2% agarose gel (Invitrogen, CA, US) electrophoresis containing SYBR Safe Dye (Jena Bioscience, Jena, DE) with visualization in transilluminator with L-Pix ultraviolet light (Loccus, SP, BR).Each sample was submitted three times to RT-PCR for each virus species tested.
Positive results were confirmed by DNA sequencing.The DNA purification was realized using ExoSAP (ThermoFisher Scientific, Waltham, MA, US) following the product protocol.Sanger sequencing (Applied Biosystems, Waltham, MA, US) was used and the chromatograms were read using the software Chromas 2.6.6 (Technelysium, South Brisbane, QLD, AU), and the DNA sequence similarity with each virus was performed using basic local alignment search tool (BLAST) from the National Center for Biotechnology Information (NCBI) (Altschul et al., 1997).The sequences were deposited in NCBI and the accession number, primer sequence, amplicon size, genomic position, and amplification target can be found in Table S2.
We evaluated the presence and absence of viruses by calculating the frequency of positive results per virus species against the total of colonies studied and per total of different stingless bee species.Positive covert frequencies were classified according to the number of viruses present in the samples into single infections, double infections, triple infections, and quadruple infections.

Results
We found out that 42.7% out of 68 samples were positive for some of the viruses studied among all seven species of stingless bees (Figure 2).Where found, the virus ABPV (present in all bee species) with 39.7% of prevalence, and IAPV (in two species) with 5.9% of prevalence.
Covert infection was observed in 7% of the positive samples which doubled virus infection for quadrifasciata anthidioides and T. angustula colonies.The prevalence of stingless bee colonies positive for viruses was greater in the winter season (52.9%) than in the summer (32.4%).ABPV virus was present in samples from both seasons (winter 47.1% and summer 32.4% of the colonies) while IAPV was only in winter (11.8%) (Figure 4).

Discussion
In this study, we report the presence of viruses ABPV and IAPV among seven Brazilian stingless bee species (Figure 2).We report the first occurrence of virus ABPV in N. testaceicornis, T. spinipes, and T. angustula, which was also the first IAPV virus-positive occurrence.Of the stingless bee species studied, T. spinipes and Partamona helleri are not bred for honey production because of their defensive behavior and the structure of their colonies, which makes it difficult to adapt in rational hives.However, these species are often sharing food sources and nesting areas with other stingless bees and also with honey bee A. mellifera.
ABPV was the most prevalent virus among our samples (Figure 2), a similar result was observed by Guimaraes-Cestaro et al. (2020) who studied viruses in stingless bees N. testaceicornis, T. angustula, and Tetragona elongata.The non-detection of many viruses surprised us since the presence of ABPV, IAPV, CBPQ, BQCV, and DWV in Apis mellifera (Peixoto et al., 2021) and DWV in Melipona spp.(De Souza et al., 2019) and Apis mellifera (Peixoto et al., 2021) have been reported in Bahia state.Therefore, virus transmission between A. mellifera and other bee species can occur as they share floral resources (Singh et al., 2010).The closer the stingless bee colonies might be to apiaries, the more likely they are to share viruses (Guzman-Novoa et al., 2015), and the meliponary where the stingless bees were sampled is only 800 m distance from apiaries in the surrounding areas.Nevertheless, even though the surrounding apiaries can be considered a hotspot for disease, spreading the virus load is important (Alger et al., 2019) and the DWV load in A. mellifera and stingless bees previously detected in the area was considered low (De Souza et al., 2019), which can explain the negative results in our samples.On the other hand, the absence of a virus from a sample cannot be extrapolated to the meliponary area or even colony, only the stingless bee sampled.
Low corvet infection was observed in our samples (Figure 2), with IAPV virus presenting lower prevalence compared to ABPV which contrasts with previous results in A. mellifera in Brazil (Figure 3) where IAPV and ABPV present more similar prevalence (Peixoto et al., 2021), therefore, the relation between viral prevalence in honey bees and wild bees can differ between viruses (Piot et al., 2022), which can explain lower viruses prevalence in wild bees, as also observed in wild bees from Argentina (Alvarez et al., 2018) andMexico (Tapia-Gonz� alez et al., 2019).
Winter was the season where IAPV and ABPV viruses were found among the species sampled (Figure 4).The climate conditions of the municipality of Cruz das Almas during the sampling period were: winter temperature of 21.5 � C, rainfall 112 mm, and  humidity of 84.7%; summer average of temperature 25.4 � C, rainfall 92.3 mm, and humidity of 78.3%.Our samples were collected within six months apart to represent dry (summer) and rainy seasons (winter), because of the size of Brazil, seasons can be different, meaning that our condition might not be similar to other areas.In our area, the temperature does not impact the plants as much as in temperate countries, which means the area will have flowers throughout the whole year.The rain, on the other hand, can be more impacting as it interferes with the bee colony harvesting activity, reducing pollen and honey storage and consequently the colony population (Do Nascimento & Nascimento, 2012).We have registered in our meliponary that the stingless bee colony populations are reduced over winter and we need to start feeding artificially (not including T. spinipes and Partamona helleri) to avoid colony loss.Therefore, the correlation between viruses, climate parameters, and vegetation in bees is registered (Piot et al., 2022).Also, the immunological system of the bees can be affected by external factors such as climatic changes and pesticides which can help the virulence driving the loss of colonies (Nazzi et al., 2012).
Studies about viruses in stingless bees are new in Brazil and we still have a long way to learn about how the pathogens are dispersed and their effect on the native bee fauna.

Conclusions
Viruses that infect A. mellifera are present in stingless bees.This is the first study in Brazil with several stingless bee species and we record the presence of ABPV in M. quadrifasciata anthidioides, M. scutellaris, Nannotrigona testaceicornis, Partamona helleri, Scaptotrigona xanthotricha, Tetragonisca angustula, and Trigona spinipes; and IAPV in M. quadrifasciata anthidioides and T. spinipes.The absence of other viruses in our study does not indicate that those viruses are not in the native stingless bees in Brazil, only that they were not detected in our samples.The study of pathogens fauna in native stingless bees is a novelty in Brazil, therefore, further studies will be necessary to evaluate and understand their dispersion among native bees and the possible impact to the bee colonies and producers.

Figure 1 .
Figure 1.Location of Municipality of Cruz das Almas, Bahia, Brazil.Where the species of native stingless bees were kept.

Figure 2 .
Figure 2. Acute bee paralysis virus (ABPV) and Israeli acute bee paralysis virus (IAPV) prevalence in seven native stingless bee species from Brazil.