Bee pathogens found in Bombus atratus from Colombia: A case study.

Bombus atratus bumblebees from Colombia that were caught in the wild and from breeding programs were screened for a broad set of bee pathogens. We discovered for the first time Lake Sinai Virus and confirmed the infection by other common viruses. The prevalence of Apicystis bombi, Crithidia bombi and Nosema ceranae was remarkably high. According to other studies the former two could have been co-introduced in South America with exotic bumble bees as Bombus terrestris or Bombus ruderatus. Given the fact that none of these species occur in Colombia, our data puts a new light on the spread of these pathogens over the South American continent.

Comprehensive Approach to Phenotype Varroa destructor Reproduction in Honey Bee Drone Brood and Its Correlation with Decreased Mite Reproduction (DMR).

The mechanisms of action behind decreased mite reproduction (DMR) are still unknown, but current hypotheses state that DMR is the result of brood-intrinsic and/or external disturbances in the V. destructor-honey bee pupa signal interactions. For accurate and precise DMR phenotyping, sufficient single infested honey bee brood cells are required (e.g., 35), which requires extensive labor and time and may exclude many samples not reaching the threshold. We defined a new comprehensive trait called the 'mean V. destructor reproduction rate' (mVR), which describes the mean number of offspring mites per infested cell in the sample while compensating for the reduced number of offspring with increasing multiple infested cells. We found a significant correlation between mVR and DMR, allowing for an estimation of DMR based on the mVR only. When the mVR was calculated with 10 infested cells, we found an average variation in mVR of 16.8%. For the same variation in DMR determination, 40 single infested cells are required. This broader look at V. destructor resistance phenotyping can improve the applicability and effectiveness of traits related to V. destructor reproduction in honey bee breeding programs.

Known and novel viruses in Belgian honey bees: yearly differences, spatial clustering, and associations with overwintering loss.

In recent years, managed honey bee colonies have been suffering from an increasing number of biotic and abiotic stressors, resulting in numerous losses of colonies worldwide. A pan-European study, EPILOBEE, estimated the colony loss in Belgium to be 32.4% in 2012 and 14.8% in 2013. In the current study, absolute viral loads of four known honey bee viruses (DWV-A, DWV-B, AmFV, and BMLV) and three novel putative honey bee viruses (Apis orthomyxovirus 1, apthili virus, and apparli virus) were determined in 300 Flemish honey bee samples, and associations with winter survival were determined. This revealed that, in addition to the known influence of DWV-A and DWV-B on colony health, one of the newly described viruses (apthili virus) shows a strong yearly difference and is also associated with winter survival. Furthermore, all scrutinized viruses revealed significant spatial clustering patterns, implying that despite the limited surface area of Flanders, local virus transmission is paramount. The vast majority of samples were positive for at least one of the seven investigated viruses, and up to 20% of samples were positive for at least one of the three novel viruses. One of those three, Apis orthomyxovirus 1, was shown to be a genuine honey bee-infecting virus, able to infect all developmental stages of the honey bee, as well as the Varroa destructor mite. These results shed light on the most prevalent viruses in Belgium and their roles in the winter survival of honey bee colonies. IMPORTANCE: The western honey bee (Apis mellifera) is a highly effective pollinator of flowering plants, including many crops, which gives honey bees an outstanding importance both ecologically and economically. Alarmingly high annual loss rates of managed honey bee colonies are a growing concern for beekeepers and scientists and have prompted a significant research effort toward bee health. Several detrimental factors have been identified, such as varroa mite infestation and disease from various bacterial and viral agents, but annual differences are often not elucidated. In this study, we utilize the viral metagenomic survey of the EPILOBEE project, a European research program for bee health, to elaborate on the most abundant bee viruses of Flanders. We complement the existing metagenomic data with absolute viral loads and their spatial and temporal distributions. Furthermore, we identify Apis orthomyxovirus 1 as a potentially emerging pathogen, as we find evidence for its active replication honey bees.

Virus Prevalence in Egg Samples Collected from Naturally Selected and Traditionally Managed Honey Bee Colonies across Europe.

Monitoring virus infections can be an important selection tool in honey bee breeding. A recent study pointed towards an association between the virus-free status of eggs and an increased virus resistance to deformed wing virus (DWV) at the colony level. In this study, eggs from both naturally surviving and traditionally managed colonies from across Europe were screened for the prevalence of different viruses. Screenings were performed using the phenotyping protocol of the 'suppressed in ovo virus infection' trait but with qPCR instead of end-point PCR and a primer set that covers all DWV genotypes. Of the 213 screened samples, 109 were infected with DWV, 54 were infected with black queen cell virus (BQCV), 3 were infected with the sacbrood virus, and 2 were infected with the acute bee paralyses virus. It was demonstrated that incidences of the vertical transmission of DWV were more frequent in naturally surviving than in traditionally managed colonies, although the virus loads in the eggs remained the same. When comparing virus infections with queen age, older queens showed significantly lower infection loads of DWV in both traditionally managed and naturally surviving colonies, as well as reduced DWV infection frequencies in traditionally managed colonies. We determined that the detection frequencies of DWV and BQCV in honey bee eggs were lower in samples obtained in the spring than in those collected in the summer, indicating that vertical transmission may be lower in spring. Together, these patterns in vertical transmission show that honey bee queens have the potential to reduce the degree of vertical transmission over time.

Polar tube protein gene diversity among Nosema ceranae strains derived from a Greek honey bee health study.

Honey bee samples from 54 apiaries originating from 37 geographic locations of Greece were screened for Nosema apis and Nosema ceranae. Furthermore 15 samples coming from 12 geographic locations were screened also for Paenibacilluslarvae and Melissococcus plutonius and seven honey bee virus species, for the first time on a nation-wide level. There was a tendency in finding proportionally higher spore counts in samples from apiaries that suffered important colony losses. P. larvae bacteria were identified in two samples and each of the tested bee viruses could be detected in at least one of the examined samples, with IAPV, CBPV and SBV being the least abundant and BQCV and DWV being the most abundant. In the study we focused on polymorphism of a N. ceranae gene encoding a polar tube protein (PTP) as similar genes were proven to be highly polymorphic in the microsporidian parasites Encephalitozoon cuniculi and Encephalitozoon hellem. The polymorphism observed in the PTP gene sequences from a single sample (bee hive) was unexpected and can thus be considered to be a major obstacle for genotyping.

An international inter-laboratory study on Nosema spp. spore detection and quantification through microscopic examination of crushed honey bee abdomens.

Nosemosis is a microsporidian disease causing mortality and weakening of honey bee colonies, especially in the event of co-exposure to other sources of stress. As a result, the disease is regulated in some countries. Reliable and harmonised diagnosis is crucial to ensure the quality of surveillance and research results. For this reason, the first European Interlaboratory Comparison (ILC) was organised in 2017 in order to assess both the methods and the results obtained by National Reference Laboratories (NRLs) in counting Nosema spp. spores by microscopy. Implementing their own routine conditions of analysis, the 23 participants were asked to perform an assay on a panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded that using microscopy to detect and quantify spores of Nosema spp. was reliable and valid.

Heritability estimates of the novel trait 'suppressed in ovo virus infection' in honey bees (Apis mellifera).

Honey bees are under pressure due to abnormal high colony death rates, especially during the winter. The infestation by the Varroa destructor mite and the viruses that this ectoparasite transmits are generally considered as the bees' most important biological threats. Almost all efforts to remedy this dual infection have so far focused on the control of the Varroa mite alone and not on the viruses it transmits. In the present study, the sanitary control of breeding queens was conducted on eggs taken from drone brood for 4 consecutive years (2015-2018). The screening was performed on the sideline of an ongoing breeding program, which allowed us to estimate the heritabilities of the virus status of the eggs. We used the term 'suppressed in ovo virus infection' (SOV) for this novel trait and found moderate heritabilities for the presence of several viruses simultaneously and for the presence of single viral species. Colonies that expressed the SOV trait seemed to be more resilient to virus infections as a whole with fewer and less severe Deformed wing virus infections in most developmental stages, especially in the male caste. The implementation of this novel trait into breeding programs is recommended.

Comparison between Apicystis cryptica sp. n. and Apicystis bombi (Arthrogregarida, Apicomplexa): Gregarine parasites that cause fat body hypertrophism in bees.

The molecular divergence, morphology and pathology of a cryptic gregarine that is related to the bee parasite Apicystis bombi Lipa and Triggiani, 1996 is described. The 18S ribosomal DNA gene sequence of the new gregarine was equally dissimilar to that of A. bombi and the closest related genus Mattesia Naville, 1930, although phylogenetic analysis supported a closer relation to A. bombi. Pronounced divergence with A. bombi was found in the ITS1 sequence (69.6% similarity) and seven protein-coding genes (nucleotide 78.05% and protein 90.2% similarity). The new gregarine was isolated from a Bombus pascuorum Scopoli, 1763 female and caused heavy hypertrophism of the fat body tissue in its host. In addition, infected cells of the hypopharyngeal gland tissue, an important excretory organ of the host, were observed. Mature oocysts were navicular in shape and contained four sporozoites, similar to A. bombi oocysts. Given these characteristics, we proposed the name Apicystis cryptica sp. n. Detections so far indicated that distribution and host species occupation of Apicystis spp. overlap at least in Europe, and that historical detections could not discriminate between them. Specific molecular assays were developed that can be implemented in future pathogen screens that aim to discriminate Apicystis spp. in bees.

Nationwide Screening for Bee Viruses and Parasites in Belgian Honey Bees.

The health of honey bees is threatened by multiple factors, including viruses and parasites. We screened 557 honey bee (Apis mellifera) colonies from 155 beekeepers distributed all over Belgium to determine the prevalence of seven widespread viruses and two parasites (Varroa sp. and Nosema sp.). Deformed wing virus B (DWV-B), black queen cell virus (BQCV), and sacbrood virus (SBV) were highly prevalent and detected by real-time RT-PCR in more than 95% of the colonies. Acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV) and deformed wing virus A (DWV-A) were prevalent to a lower extent (between 18 and 29%). Most viruses were only present at low or moderate viral loads. Nevertheless, about 50% of the colonies harbored at least one virus at high viral load (>107 genome copies/bee). Varroa mites and Nosema sp. were found in 81.5% and 59.7% of the honey bee colonies, respectively, and all Nosema were identified as Nosema ceranae by real time PCR. Interestingly, we found a significant correlation between the number of Varroa mites and DWV-B viral load. To determine the combined effect of these and other factors on honey bee health in Belgium, a follow up of colonies over multiple years is necessary.

Ontogeny of the chondrocranium in Corydoras aeneus (Gill, 1858) (Callichthyidae, Siluriformes).

Callichthyids take a basal position in the loricarioid evolutionary lineage leading up to an algae scraping feeding mechanism in the loricariid family. Therefore, the study of the morphology and development of a callichthyid representative would contribute to a better knowledge on the differences in cranial morphology and their impact on feeding ecology within this superfamily. Therefore, development in the chondrocranium of Corydoras aeneus was studied based on 22 cleared and stained specimens and 6 series of serial sections. The latter sections were also digitized and used for 3D reconstructions. Development overall follows the typical siluriform trends in chondrocranial development. Even the low complexity of the chondrocranium at hatching fits the trend observed in other siluriforms, although other studies showed loricarioid hatchlings to generally show more complex chondrocrania. In contrast to other catfish, in C. aeneus, the notochord was never found to protrude into the hypophyseal fenestra. In addition, also differing from other siluriforms, a commissura lateralis is present, a state also reported for Ancistrus cf. triradiatus (Geerinckx et al., [2005] J Morphol 266:331-355). The splanchnocranium again has the typical siluriform shape during its ontogeny, with the presence of a compound hyosymplectic-pterygoquadrate plate, although not fused to the neurocranium or interhyal at any time during ontogeny, a state described earlier for Callichthys callichthys (Hoedeman, [1960a] Bull Aquat Biol 1:73-84; Howes and Teugels, [1989] J Zool Lond 219:441-456). The most striking difference found in comparison to other catfishes, however, involves thebranchial basket, which arises as a single element with a further differentiation from the middle arches on in both a rostral and caudal direction.

Development of the osteocranium in the suckermouth armored catfish Ancistrus cf. triradiatus (Loricariidae, siluriformes).

The development of the osteocranium of the suckermouth armored catfish Ancistrus cf. triradiatus is described based on specimens ranging from prehatching stages to juvenile stages where the osteocranium is more or less fully formed. The first bony elements that arise are the opercle, jaws, and lateralmost branchiostegal rays, as well as the basioccipital and parasphenoid in the skull floor. The supracleithrum and the membranous and perichondral pterotic components form one large, double-layered skull bone during ontogeny, without clear evidence of the involvement of a supratemporal. The Baudelot's ligament ossifies from two sides, i.e., from the basioccipital medially and the supracleithrum laterally. The lower jaw consists of a dentary, mentomeckelian, and angulo-articular, which all soon fuse. The parurohyal, formed by the fusion of a ventral sesamoid bone and a dorsal cartilage element associated with the first basibranchial, is pierced by a vein, unlike in some other siluriforms. The interhyal cartilage disappears during ontogeny; medially of it, a small sesamoid bone appears in a ligament. The largest, canal-bearing cheek plate is not homologous to the interopercle. The results of the present research, with emphasis on bone formations and homologies, are compared with studies on related catfishes.

Nosema neumanni n. sp. (Microsporidia, Nosematidae), a new microsporidian parasite of honeybees, Apis mellifera in Uganda.

The microsporidium Nosema neumanni n. sp., a new parasite of the honeybee Apis mellifera is described based on its ultra-structural and molecular characteristics. Structures resembling microsporidian spores were found by microscopic examination of honeybees from Uganda. Molecular confirmation failed when PCR primers specific for Nosema apis and Nosema ceranae were used, but was successful with primers covering the whole family of Nosematidae. We performed transmission electron microscopy and found typical microsporidian spores which were smaller (length: 2.36±0.14µm and width: 1.78±0.06µm; n=6) and had fewer polar filament coils (10-12) when compared to those of known species infecting honeybees. The entire 16S SSU rRNA region was amplified, cloned and sequenced and was found to be unique with the highest resemblance (97% identity) to N. apis. The incidence of N. neumanni n. sp. in Ugandan honeybees was found to be much higher than of the two other Nosema species.

Molecular cloning and expression of icarapin, a novel IgE-binding bee venom protein.

The 1045bp full-length cDNA sequence of a new bee venom component was obtained by rapid amplification of cDNA ends. The 672bp coding sequence corresponds to a protein with a signal peptide and multiple carbohydrate binding sites, and it was named icarapin. It has the new consensus sequence N-[TS]-T-S-[TV]-x-K-[VI](2)-[DN]-G-H-x-V-x-I-N-[ED]-T-x-Y-x-[DHK]-x(2,6)- [STA]-[VLFI]-x-[KR]-V-R-[VLI]-[IV]-[DN]-V-x-P. At least two transcript variants were found. Recombinant icarapin was tested for recognition by IgE antibodies and gave a positive dot blot with sera from 4 out of 5 bee venom allergic patients, all beekeepers. Indirect immunofluorescent staining localized the protein in the cuticular lining of the venom duct.

First detection of Paenibacillus larvae the causative agent of American Foulbrood in a Ugandan honeybee colony.

Paenibacillus larvae is a highly contagious and often lethal widely distributed pathogen of honeybees, Apis mellifera but has not been reported in eastern Africa to date. We investigated the presence of P. larvae in the eastern and western highland agro-ecological zones of Uganda by collecting brood and honey samples from 67 honeybee colonies in two sampling occasions and cultivated them for P. larvae. Also, 8 honeys imported and locally retailed in Uganda were sampled and cultivated for P. larvae. Our aim was to establish the presence and distribution of P. larvae in honeybee populations in the two highland agro-ecological zones of Uganda and to determine if honeys that were locally retailed contained this lethal pathogen. One honeybee colony without clinical symptoms for P. larvae in an apiary located in a protected area of the western highlands of Uganda was found positive for P. larvae. The strain of this P. larvae was genotyped and found to be ERIC I. In order to compare its virulence with P. larvae reference strains, in vitro infection experiments were conducted with carniolan honeybee larvae from the research laboratory at Ghent University, Belgium. The results show that the virulence of the P. larvae strain found in Uganda was at least equally high. The epidemiological implication of the presence of P. larvae in a protected area is discussed.

Development of the chondrocranium in the suckermouth armored catfish Ancistrus cf. triradiatus (Loricariidae, Siluriformes).

The chondrocranium of the suckermouth armored catfish Ancistrus cf. triradiatus was studied. Its development is described based on specimens ranging from small prehatching stages with no cartilage visible, to larger posthatching stages where the chondrocranium is reducing. Cleared and stained specimens, as well as serial sections, revealed a cartilaginous skeleton with many features common for Siluriformes, yet several aspects of A. cf. triradiatus are not seen as such in other catfishes, or to a lesser extent. The skull is platybasic, but the acrochordal cartilage is very small and variably present, leaving the notochord protruding into the hypophyseal fenestra in the earlier stages. The ethmoid region is slender, with a rudimentary solum nasi. A lateral commissure and myodomes are present. The larger posterior myodome is roofed by a prootic bridge. The maxillary barbel is supported by a conspicuous cartilaginous rod from early prehatching stages. The ceratohyal has four prominent lateral processes. Infrapharyngobranchials I-II do not develop. During ontogeny, the skull lengthens, with an elongated ethmoid, pointing ventrally, and a long and bar-shaped hyosymplectic-pterygoquadrate plate. Meckel's cartilages point medially instead of rostrally.

Development of the osteocranium in Corydoras aeneus (Gill, 1858) Callichthyidae, Siluriformes.

Development in the osteocranium of Corydoras aeneus was studied based on 48 cleared and stained specimens and 10 series of serial sections. Development overall follows the general trends observed in siluriform development, with ossifications appearing as a response to functional demands. Early development of the skull occurs in two distinct phases. In a first phase, several new bony elements, all of dermal origin and related to feeding, appear shortly after yolk depletion (4.4 mm SL). Between 5 and 8 mm SL, developmental priorities seem to shift to size increase of the cartilaginous skull and no new bony elements appear. Finally, a second phase of osteogenesis occurs from 8 to 18 mm SL, in which all remaining dermal and perichondral bones appear.

Cranial architecture of tube-snouted gasterosteiformes (Syngnathus rostellatus and Hippocampus capensis).

The long snout of pipefishes and seahorses (Syngnathidae, Gasterosteiformes) is formed as an elongation of the ethmoid region. This is in contrast to many other teleosts with elongate snouts (e.g., butterflyfishes) in which the snout is formed as an extension of the jaws. Syngnathid fishes perform very fast suction feeding, accomplished by powerful neurocranial elevation and hyoid retraction. Clearly, suction through a long and narrow tube and its hydrodynamic implications can be expected to require certain adaptations in the cranium, especially in musculoskeletal elements of the feeding apparatus. Not much is known about which skeletal elements actually support the snout and what the effect of elongation is on related structures. Here, we give a detailed morphological description of the cartilaginous and bony feeding apparatus in both juvenile and adult Syngnathus rostellatus and Hippocampus capensis. Our results are compared with previous morphological studies of a generalized teleost, Gasterosteus aculeatus. We found that the ethmoid region is elongated early during development, with the ethmoid plate, the hyosymplectic, and the basihyal cartilage being extended in the chondrocranium. In the juveniles of both species almost all bones are forming, although only as a very thin layer. The elongation of the vomeral, mesethmoid, quadrate, metapterygoid, symplectic, and preopercular bones is already present. Probably, because of the long and specialized parental care which releases advanced developmental stages from the brooding pouch, morphology of the feeding apparatus of juveniles is already very similar to that of the adults. We describe morphological features related to snout elongation that may be considered adaptations for suction feeding; e.g. the peculiar shape of the interhyal bone and its saddle-shaped articulation with the posterior ceratohyal bone might aid in explosive hyoid retraction by reducing the risk of hyoid dislocation.

Functional and evolutionary insights from the genomes of three parasitoid Nasonia species.

We report here genome sequences and comparative analyses of three closely related parasitoid wasps: Nasonia vitripennis, N. giraulti, and N. longicornis. Parasitoids are important regulators of arthropod populations, including major agricultural pests and disease vectors, and Nasonia is an emerging genetic model, particularly for evolutionary and developmental genetics. Key findings include the identification of a functional DNA methylation tool kit; hymenopteran-specific genes including diverse venoms; lateral gene transfers among Pox viruses, Wolbachia, and Nasonia; and the rapid evolution of genes involved in nuclear-mitochondrial interactions that are implicated in speciation. Newly developed genome resources advance Nasonia for genetic research, accelerate mapping and cloning of quantitative trait loci, and will ultimately provide tools and knowledge for further increasing the utility of parasitoids as pest insect-control agents.