Detection of Apicystis bombi (Apicomplexa: Neogregarinorida) in carpenter bees of Argentina.

Historically, the neogregarine Apicystis bombi was isolated almost exclusively from bumble bees (Bombus spp.) where it disrupts adipose tissue, increasing hosts' mortality rates. Records in solitary bees are scarce worldwide. To check for its presence in carpenter bees (genus Xylocopa), campaigns were performed in Argentina capturing 154 individuals of five species (X. augusti, X. splendidula, X. atamisquensis, X. frontalis, X. nigrocincta). The presence of A. bombi was detected by molecular means in X. augusti, X. atamisquensis, and X. nigrocincta in four of the nine provinces screened. The pathogenesis and eventual impact that A. bombi may cause in individuals or populations of Xylocopa species remain unknown. The presence of A. bombi in northern Argentina would be contradictory to the hypothesis that its occurrence is the exclusive result of its introduction to South America through invasive, infected exotic bumble bees.

Parasites and RNA viruses in wild and laboratory reared bumble bees Bombus pauloensis (Hymenoptera: Apidae) from Uruguay.

Bumble bees (Bombus spp.) are important pollinators insects involved in the maintenance of natural ecosystems and food production. Bombus pauloensis is a widely distributed species in South America, that recently began to be managed and commercialized in this region. The movement of colonies within or between countries may favor the dissemination of parasites and pathogens, putting into risk while populations of B. pauloensis and other native species. In this study, wild B. pauloensis queens and workers, and laboratory reared workers were screened for the presence of phoretic mites, internal parasites (microsporidia, protists, nematodes and parasitoids) and RNA viruses (Black queen cell virus (BQCV), Deformed wing virus (DWV), Acute paralysis virus (ABCV) and Sacbrood virus (SBV)). Bumble bee queens showed the highest number of mite species, and it was the only group where Conopidae and S. bombi were detected. In the case of microsporidia, a higher prevalence of N. ceranae was detected in field workers. Finally, the bumble bees presented the four RNA viruses studied for A. mellifera, in proportions similar to those previously reported in this species. Those results highlight the risks of spillover among the different species of pollinators.

Lotmaria passim (Kinetoplastea: Trypanosomatidae) in honey bees from Argentina.

Lotmaria passim (Kinetoplastea) is considered the most prevalent as well as the most virulent trypanosomatid associated to the European honey bee Apis mellifera. We used qPCR to screen for the presence of this parasite in 57 samples from ten Argentinian provinces, and were able to detect its presence throughout most of the country with 41% of the samples testing positive. In a retrospective analysis, we detected L. passim in 73% of honey bee samples from 2006 showing that this flagellate has been widely present in Argentina for at least ~15 years. Additionally, three primer sets for L. passim detection were compared, with the pair that produced smallest PCR product having the best detection capability. Finally, we also found L. passim DNA in 100% (n = 6) of samples of the mite Varroa destructor. The role of this ectoparasite in the lifecycle of Lotmaria, if any, remains unrevealed.

Chronic bee paralysis virus (CBPV) in South American non-Apis bees.

Chronic bee paralysis virus (CBPV) is a positive single-stranded RNA virus that exhibits a worldwide distribution. Although the effects of this virus on honeybees' health are well known, its presence in other bee species has not been fully studied. In this work, CBPV was detected in several native bees from Argentina, including Bombus pauloensis, Halictillus amplilobus, Peponapis fervens, and members of the genus Xylocopa. Here, we report for the first time the presence of CBPV in native bees from South America.

Long-term prevalence of the protists Crithidia bombi and Apicystis bombi and detection of the microsporidium Nosema bombi in invasive bumble bees.

An initial survey in 2009 carried out at a site in northwestern Patagonia region, Argentina, revealed for the first time in South America the presence of the flagellate Crithidia bombi and the neogregarine Apicystis bombi, two pathogens associated with the Palaearctic invasive bumble bee Bombus terrestris. In order to determine the long-term persistence and dynamics of this microparasite complex, four additional collections at the same site (San Carlos de Bariloche) were conducted along the following seven years. Both protists were detected in all collections: prevalence was 2%-21.6% for C. bombi and 1.2%-14% for A. bombi. In addition, the microsporidium Nosema bombi was recorded for the first time in the country in the last two collections, at prevalences of 12.4% and 2.4% and unusually high infection intensities (Average = 6.56 × 107 spores per individual). Due to the exceptional dispersal ability of the exotic B. terrestris, these three multihost pathogens should be considered as potential threats to South American native bumble bees.

Tubulinosema pampeana sp. n. (Microsporidia, Tubulinosematidae), a pathogen of the South American bumble bee Bombus atratus.

An undescribed microsporidium was detected and isolated from the South American bumble bee Bombus atratus collected in the Pampas region of Argentina. Infection intensity in workers averaged 8.2 × 10(7)spores/bee. The main site of infection was adipose tissue where hypertrophy of adipocytes resulted in cyst-like body formation. Mature spores were ovoid and monomorphic. They measured 4.00 µm × 2.37 µm (fresh) or 3.98 µm × 1.88 µm (fixed). All stages were diplokariotic and developed in direct contact with host cytoplasm. Isofilar polar filament was arranged in 16 coils in one or, posteriorly, two layers. Coiling angle was variable, between perpendicular and almost parallel to major spore axis. Late meronts and sporogonial stages were surrounded by vesicles of approximately 60 nm in diameter. Based on both new and already designed primers, a 1827 bp (SSUrRNA, ITS, LSUrRNA) sequence was obtained. Data analyses suggest that this microsporidium is a new species of the genus Tubulinosema. The name Tubulinosema pampeana sp. n. is proposed.

The invasion of southern South America by imported bumblebees and associated parasites.

The Palaearctic Bombus ruderatus (in 1982/1983) and Bombus terrestris (1998) have both been introduced into South America (Chile) for pollination purposes. We here report on the results of sampling campaigns in 2004, and 2010-2012 showing that both species have established and massively expanded their range. Bombus terrestris, in particular, has spread by some 200 km year(-1) and had reached the Atlantic coast in Argentina by the end of 2011. Both species, and especially B. terrestris, are infected by protozoan parasites that seem to spread along with the imported hosts and spillover to native species. Genetic analyses by polymorphic microsatellite loci suggest that the host population of B. terrestris is genetically diverse, as expected from a large invading founder population, and structured through isolation by distance. Genetically, the populations of the trypanosomatid parasite, Crithidia bombi, sampled in 2004 are less diverse, and distinct from the ones sampled later. Current C. bombi populations are highly heterozygous and also structured through isolation by distance correlating with the genetic distances of B. terrestris, suggesting the latter's expansion to be a main structuring factor for the parasite. Remarkably, wherever B. terrestris spreads, the native Bombus dahlbomii disappears although the reasons remain unclear. Our ecological and genetic data suggest a major invasion event that is currently unfolding in southern South America with disastrous consequences for the native bumblebee species.

Genetic variability of the neogregarine Apicystis bombi, an etiological agent of an emergent bumblebee disease.

The worldwide spread of diseases is considered a major threat to biodiversity and a possible driver of the decline of pollinator populations, particularly when novel species or strains of parasites emerge. Previous studies have suggested that populations of introduced European honeybee (Apis mellifera) and bumblebee species (Bombus terrestris and Bombus ruderatus) in Argentina share the neogregarine parasite Apicystis bombi with the native bumblebee (Bombus dahlbomii). In this study we investigated whether A. bombi is acting as an emergent parasite in the non-native populations. Specifically, we asked whether A. bombi, recently identified in Argentina, was introduced by European, non-native bees. Using ITS1 and ITS2 to assess the parasite's intraspecific genetic variation in bees from Argentina and Europe, we found a largely unstructured parasite population, with only 15% of the genetic variation being explained by geographic location. The most abundant haplotype in Argentina (found in all 9 specimens of non-native species) was identical to the most abundant haplotype in Europe (found in 6 out of 8 specimens). Similarly, there was no evidence of structuring by host species, with this factor explaining only 17% of the genetic variation. Interestingly, parasites in native Bombus ephippiatus from Mexico were genetically distant from the Argentine and European samples, suggesting that sufficient variability does exist in the ITS region to identify continent-level genetic structure in the parasite. Thus, the data suggest that A. bombi from Argentina and Europe share a common, relatively recent origin. Although our data did not provide information on the direction of transfer, the absence of genetic structure across space and host species suggests that A. bombi may be acting as an emergent infectious disease across bee taxa and continents.

Effects of the organic acids produced by a lactic acid bacterium in Apis mellifera colony development, Nosema ceranae control and fumagillin efficiency.

The European honey bee Apis mellifera is known to be affected by many parasites and pathogens that have great impact over the insect development. Among parasites affecting bee health, Nosema ceranae is one of the main biotic factors affecting colony populations. As honey bee populations decline, interest in pathogenic and mutualistic relationships between bees and microorganisms has increased. The main goal of the current study was to assess the effect of the oral administration of the metabolites produced by Lactobacillus johnsonii CRL1647 (mainly organic acids) supplemented in syrup, on: (I) N. ceranae sporulation dynamics before and after fumagillin application, and (II) performance of A. mellifera colonies. Different experiments were conducted to evaluate the effects of these bacterial metabolites on bees: in vitro administration revealed no toxic effects against bees. Colonies fed with the lactic acids incremented their beehive population and also the amount of fat bodies per bee. Finally, the organic acids reduced the intensity of the pathogen after the second application of treatment as well as enhanced the fumagillin efficiency. This study provides important information for the development of new control substances against nosemosis.

The tracheal mite Locustacarus buchneri in South American native bumble bees (Hymenoptera: Apidae).

As in other regions of the world, bumble bees (Bombus spp.) are important pollinators in the neotropics. Despite its relevance, knowledge on their health is still limited in the region. While external acari are known to occur in these insects, presence of the internal, tracheal mite Locustacarus buchneri is here reported for first time. After the examination of 2,508 individuals of eight Bombus species from Argentina, two workers of Bombus bellicosus and one of Bombus atratus were found parasitized by L. buchneri in localities within San Luis and Buenos Aires provinces, respectively. The rare occurrence recorded agrees with findings from elsewhere in the world.

Spore loads of Paranosema locustae (Microsporidia) in heavily infected grasshoppers (Orthoptera: Acridoidea) of the Argentine Pampas and Patagonia.

Paranosema locustae, an entomopathogen of grasshoppers and locusts, remains the only microsporidium registered as a biocontrol agent. After introductions from North America, it became established in grasshopper communities of Argentina. We measured the infection intensity of field collected, heavily infected male and female adults of individuals belonging to six grasshopper species, five melanoplines (Melanoplinae) (Baeacris pseudopunctulatus, Dichroplus maculipennis, Dichroplus vittatus, Neopedies brunneri, Scotussa lemniscata), and one gomphocerine (Gomphocerinae) (Staurorhectus longicornis). Average spore load among heavily infected grasshoppers ranged from 8.7±0.5×10(7) to 1.1±0.7×10(9). Only females of B. pseudopunctulatus and S. longicornis showed significantly higher spore loads than the males.

Sphaerularia bombi (Nematoda: Sphaerulariidae) parasitizing Bombus atratus (Hymenoptera: Apidae) in southern South America.

Bumble bees are some of the most important insect pollinators. However, knowledge on parasites associated to bumble bees in South America is very limited. This study reports the first isolation of a sphaerularid nematode parasitizing queens of the native bumble bee Bombus atratus in Argentina. Measurements and morphological characters of eggs, juveniles, and adults strongly suggest that the species is Sphaerularia bombi, a parasite that affects the reproduction and foraging behavior of the host. The nematode was detected in bumble bees of San Carlos de Bariloche, northwestern Patagonia region, and the surroundings of La Plata, northeastern Pampas region. Prevalence varied between 8% and 20%.

Apicystis bombi (Apicomplexa: Neogregarinorida) parasitizing Apis mellifera and Bombus terrestris (Hymenoptera: Apidae) in Argentina.

The neogregarine Apicystis bombi is considered a low prevalence parasite of Bombus spp. Before our work it has only once been detected in one single specimen of the Western honeybee Apis mellifera. This contribution reports the presence of A. bombi parasitizing both A. mellifera and Bombus terrestris at a site in Northwestern Argentine Patagonia (Bariloche, close to the border with Chile) and analyses its possible absence in the Pampas region, the most important beekeeping region of the country. In Bariloche, prevalence of A. bombi in A. mellifera was 7.6% in 2009, and 13.6% in 2010, whereas in B. terrestris it was 12.1%. Infections were not detected in 302 bee hives periodically prospected along 3 years (almost 400 000 honeybee specimens) in the Pampas. Analysis with the probability program FreeCalc2 suggested a possible absence of A. bombi in this area. Because of high virulence showed in several species of Bombus in the Northern hemisphere, A. bombi should be closely monitored in A. mellifera and in native Bombus species or other Apidae.

Invasive Bombus terrestris (Hymenoptera: Apidae) parasitized by a flagellate (Euglenozoa: Kinetoplastea) and a neogregarine (Apicomplexa: Neogregarinorida).

The flagellate Crithidia bombi and the neogregarine Apicystis bombi have been found in individuals of Bombus terrestris, a Palaearctic species of bumble bee commercially reared and shipped worldwide for pollination services. B. terrestris has recently entered into the northwestern Patagonia region of Argentina from Chile, where it was introduced in 1998. Prevalence was 21.6% for C. bombi and 3.6% for A. bombi (n=111). The pathogens were not detected in 441 bumble bees belonging to five of the eight known Argentine native species (Bombus atratus, Bombus morio, Bombus bellicosus, Bombus opifex, Bombus tucumanus) collected elsewhere in the country. Although the absence of natural occurrence of C. bombi and A. bombi in Argentine native bumble bees cannot be ascertained at present due to the limited surveys performed, it is important to report their detection in invasive B. terrestris. The invasion event is relatively recent and the accompanying pathogens are not species specific within the genus Bombus.

South American native bumblebees (Hymenoptera: Apidae) infected by Nosema ceranae (Microsporidia), an emerging pathogen of honeybees (Apis mellifera).

As pollination is a critical process in both human-managed and natural terrestrial ecosystems, pollinators provide essential services to both nature and humans. Pollination is mainly due to the action of different insects, such as the bumblebee and the honeybee. These important ecological and economic roles have led to widespread concern over the recent decline in pollinator populations that has been detected in many regions of the world. While this decline has been attributed in some cases to changes in the use of agricultural land, the effects of parasites could play a significant role in the reduction of these populations. For the first time, we describe here the presence of Nosema ceranae, an emerging honeybee pathogen, in three species of Argentine native bumblebees. A total of 455 bumblebees belonging to six species of genus Bombus were examined. PCR results showed that three of the species are positive to N. ceranae (Bombus atratus, Bombus morio and Bombus bellicosus). We discuss the appearance of this pathogen in the context of the population decline of this pollinators.