Geographical sampling bias on the assessment of endemism areas for marine meiobenthic fauna.

Species distribution patterns are constrained by historical and ecological processes in space and time, but very often the species range sizes are geographical sampling biases resulting from unequal sampling effort. One of the most common definitions of endemism is based on the "congruence of distributional areas" criterion, when two or more species have the same distributional limits. By acknowledging that available data of marine meiobenthic species are prone to geographical sampling bias and that can affect the accuracy of the biogeographical signals, the present study combines analyses of inventory incompleteness and recognition of spatial congruence of Gastrotricha, Kinorhyncha, meiobenthic Annelida and Tardigrada in order to better understand the large-scale distribution of these organisms in coastal and shelf areas of the world. We used the marine bioregionalization framework for geographical operative units to quantify the inventory incompleteness effect (by modelling spatial predictions of species richness) and to recognize areas of endemism. Our models showed that the difference between observed and expected species richness in the Southern Hemisphere is much higher than in the Northern Hemisphere. Parsimony Analysis of Endemicity delimited 20 areas of endemism, most found in the Northern Hemisphere. Distribution patterns of meiobenthic species are shown to respond to events of geographical barriers and abiotic features, and their distribution is far from homogeneous throughout the world. Also, our data show that ecoregions with distinct biotas have at least some cohesion over evolutionary time. However, we found that inventory incompleteness may significantly affect the explanatory power of areas of endemism delimitation in both hemispheres. Yet, whereas future increases in sampling efforts are likely to change the spatial congruence ranges in the Southern Hemisphere, patterns for the Northern Hemisphere may prove to be relatively more resilient.

The nuclear and mitochondrial genomes of Frieseomelitta varia - a highly eusocial stingless bee (Meliponini) with a permanently sterile worker caste.

BACKGROUND: Most of our understanding on the social behavior and genomics of bees and other social insects is centered on the Western honey bee, Apis mellifera. The genus Apis, however, is a highly derived branch comprising less than a dozen species, four of which genomically characterized. In contrast, for the equally highly eusocial, yet taxonomically and biologically more diverse Meliponini, a full genome sequence was so far available for a single Melipona species only. We present here the genome sequence of Frieseomelitta varia, a stingless bee that has, as a peculiarity, a completely sterile worker caste. RESULTS: The assembly of 243,974,526 high quality Illumina reads resulted in a predicted assembled genome size of 275 Mb composed of 2173 scaffolds. A BUSCO analysis for the 10,526 predicted genes showed that these represent 96.6% of the expected hymenopteran orthologs. We also predicted 169,371 repetitive genomic components, 2083 putative transposable elements, and 1946 genes for non-coding RNAs, largely long non-coding RNAs. The mitochondrial genome comprises 15,144 bp, encoding 13 proteins, 22 tRNAs and 2 rRNAs. We observed considerable rearrangement in the mitochondrial gene order compared to other bees. For an in-depth analysis of genes related to social biology, we manually checked the annotations for 533 automatically predicted gene models, including 127 genes related to reproductive processes, 104 to development, and 174 immunity-related genes. We also performed specific searches for genes containing transcription factor domains and genes related to neurogenesis and chemosensory communication. CONCLUSIONS: The total genome size for F. varia is similar to the sequenced genomes of other bees. Using specific prediction methods, we identified a large number of repetitive genome components and long non-coding RNAs, which could provide the molecular basis for gene regulatory plasticity, including worker reproduction. The remarkable reshuffling in gene order in the mitochondrial genome suggests that stingless bees may be a hotspot for mtDNA evolution. Hence, while being just the second stingless bee genome sequenced, we expect that subsequent targeting of a selected set of species from this diverse clade of highly eusocial bees will reveal relevant evolutionary signals and trends related to eusociality in these important pollinators.

European Foulbrood in stingless bees (Apidae: Meliponini) in Brazil: Old disease, renewed threat.

Stingless bees (Apidae: Meliponini) are a group of bees with vestigial stings showing a high level of social organization. They are important pollinators in tropical and subtropical regions, and, in the last decades, stingless beekeeping has increased rapidly in Brazil. Bee-collected pollen and honey of Apis mellifera can be an important source of disease when used as supplements to feed stingless bee colonies, a common and increasing practice adopted by stingless beekeepers. Here, we aimed to investigate the presence of pathogens commonly found in honey bees in diseased colonies of Melipona species in Espírito Santo and São Paulo States, Southeast Brazil. We detected, for the first time, the bacterium Melissococcus plutonius and symptoms of European foulbrood in Melipona spp., associated with brood death and colony losses in some cases. In addition, we tested for the presence of the bacterium Paenibacillus larvae and the fungus Aschosphaera apis, as well as the six more common honey bee viruses in Brazil (BQCV, ABPV, DWV, KBV, IAPV, CBPV) and the microsporidia Nosema apis and Nosema ceranae. However, only one sample of brood was infected with N. ceranae and all other pathogens, with the exception of Melissococcus plutonius, were absent in the analyzed brood. Lastly, we looked for toxic pollen in all food fed to diseased colonies, but none was present.

The Orchid Bee Fauna (Hymenoptera: Apidae: Euglossini) of a Neotropical Savanna: an Efficient Protocol to Assess Bee Community and Diversity Along Elevational and Habitat Complexity Gradients.

Males of euglossine bees, also known as orchid bees, are attracted to aromatic compounds and this feature has been exploited to collect them. Here we sampled orchid bee males using a rapid passive method in a Neotropical savanna ecosystem in Rio Preto State Park (PERP), southeastern Brazil. The sampling protocol consisted of 5-day surveys using scent traps at the beginning and end of the rainy seasons. PERP is inserted in a Brazilian savanna characterized by a mosaic of vegetation types, and elevational gradients. We also analyzed whether the habitat complexity and elevational gradients influence the richness and abundance of these bees. We collected a total of 996 individuals belonging to 14 species and four genera (Eufriesea Cockerell 1908, Euglossa Latreille 1802, Eulaema Lepeletier 1841, and Exaerete Hoffmannsegg 1817). Comparing our results with previous sampling protocols at PERP, higher abundance and richness were observed using traps than insect nets to survey these bees. In addition, the orchid bee fauna in PERP was successfully assessed by our rapid passive protocol, showing that this protocol seems to be useful for collecting orchid bees in open vegetation environments. We observed that the abundance of bees was lower at higher elevations, and both abundance and richness increased in more complex habitat. This may be due to the characteristics of Brazilian savannas where the lower elevations have higher habitat complexity, with more places for nest construction and resources for feeding and nesting.

Immunity and physiological changes in adult honey bees (Apis mellifera) infected with Nosema ceranae: The natural colony environment.

Nosema ceranae is a microsporidium that infects Apis mellifera, causing diverse physiological and behavioral alterations. Given the existence of individual and social mechanisms to reduce infection and fungal spread in the colony, bees may respond differently to infection depending on their rearing conditions. In this study, we investigated the effect of N. ceranae in honey bee foragers naturally infected with different fungal loads in a tropical region. In addition, we explored the effects of N. ceranae artificially infected young bees placed in a healthy colony under field conditions. Honey bees naturally infected with higher loads of N. ceranae showed downregulation of genes from Toll and IMD immune pathways and antimicrobial peptide (AMP) genes, but hemolymph total protein amount and Vitellogenin (Vg) titers were not affected. Artificially infected bees spread N. ceranae to the controls in the colony, but fungal loads were generally lower than those observed in cages, probably because of social immunity. Although no significant changes in mRNA levels of AMP-encoding were observed, N. ceranae artificially infected bees showed downregulation of miR-989 (an immune-related microRNA), lower vitellogenin gene expression, and decreased hemolymph Vg titers. Our results demonstrate for the first time that natural infection by N. ceranae suppresses the immune system of honey bee foragers in the field. This parasite is detrimental to the immune system of young and old bees, and disease spread, mitigation and containment will depend on the colony environment.

Trade-off between immune stimulation and expression of storage protein genes.

Proteins stored in insect hemolymph may serve as a source of amino acids and energy for metabolism and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCR and Western blot. After ensuring that the immune system had been activated by measuring the ensuing expression of the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (proPO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLp-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLp-III transcripts. Our findings are consistent with a down-regulation of the expression and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon represents a strategy to redirect resources to combat injury or infection.

Integrative taxonomy of a new Redudasys species (Gastrotricha: Macrodasyida) sheds light on the invasion of fresh water habitats by macrodasyids.

The order Macrodasyida (Gastrotricha) includes over 350 marine species, and only 3 freshwater species (Marinellina flagellata, Redudasys fornerise, R. neotemperatus). Herein we describe a new freshwater species of Macrodasyida, Redudasys brasiliensis sp. nov., from Brazil through an integrative taxonomic approach. The external morphology and internal anatomy were investigated using differential interference contrast microscopy, confocal microscopy, scanning and transmission electron microscopy. The systematization of the new taxon was inferred by nuclear (18S and 28S) and mitochondrial (COI) genes, and its intra-order relationships were assessed using data from most of available macrodasyids. Phylogenetic analyses yielded congruent trees, in which the new taxon is nested within the family Redudasyidae, but it was genetically distinct from the other species of the genus Redudasys. The new species shares the gross morphology and reproductive traits with other Redudasyidae and the presence of only 1 anterior adhesive tube per side with Redudasys neotemperatus, but it has a specific pattern of ventral ciliation and muscle organization. Results support the hypothesis that dispersion into fresh water habitats by Macrodasyida and Chaetonotida taxa occurred independently and that within Macrodasyida a single lineage invaded the freshwater environment only once. Furthermore, the Neotropical region seems to be peculiar for the evolution of the freshwater macrodasyid clade.

Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in three stingless bee species (Hymenoptera: Apidae: Meliponini).

Stingless bees are generalist pollinators distributed through the pantropical region. There is growing evidence that their wild populations are experiencing substantial decline in response to habitat degradation and pesticides. Policies for conservation of endangered species will benefit from studies focusing on genetic and molecular aspects of their development and behavior. The most common method for looking at gene expression is real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR) of the mRNA of interest. This method requires the identification of reliable reference genes to correctly estimate fluctuations in transcript levels. To contribute to molecular studies on stingless bees, we used Frieseomelitta varia, Melipona quadrifasciata, and Scaptotrigona bipunctata species to test the expression stability of eight reference genes (act, ef1-α, gapdh, rpl32, rps5, rps18, tbp, and tbp-af) in RT-qPCR procedures in five physiological and experimental conditions (development, sex, tissues, bacteria injection, and pesticide exposure). In general, the rpl32, rps5 and rps18 ribosomal protein genes and tpb-af gene showed the highest stability, thus being identified as suitable reference genes for the three stingless bee species and defined conditions. Our results also emphasized the need to evaluate the stability of candidate genes for any designed experimental condition and stingless bee species.

Immunosenescence in honey bees (Apis mellifera L.) is caused by intrinsic senescence and behavioral physiology.

Young honey bee workers (0 to 2-3 weeks old) perform tasks inside the colony, including brood care (nursing), whereas older workers undergo foraging tasks during the next 3-4 weeks, when an intrinsic senescence program culminates in worker death. We hypothesized that foragers are less able to react to immune system stimulation than nurse bees and that this difference is due to an inefficient immune response in foragers. To test this hypothesis, we used an experimental design that allowed us to uncouple chronological age and behavior status (nursing/foraging). Worker bees from a normal age demography colony (where workers naturally transit from nursing to foraging tasks as they age) and of a single-cohort colony setup (composed of same-aged workers performing nursing or foraging tasks) were tested for survival and capability of activation of the immune system after bacterial injection. Expression of an antimicrobial peptide gene, defensin-1 (def-1), was used to assess immune system activation. We then checked whether the immune response includes changes in the expression of aging- and behavior-related genes, specifically vitellogenin (vg), juvenile hormone esterase (jhe), and insulin-like peptide-1 (ilp-1). We found a significant difference in survival rate between bees of different ages but carrying out the same tasks. Our results thus indicate that the bees' immune response is negatively affected by intrinsic senescence. Additionally, independent of age, foragers had a shorter lifespan than nurses after bacterial infection, although both were able to induce def-1 transcription. In the normal age demography colony, the immune system activation resulted in a reduction in the expression of vg, jhe and ilp-1 genes in foragers, but not in the nurse bees, demonstrating that age and behavior are both important influences on the bees' immune response. By disentangling the effects of age and behavior in the single-cohort colony, we found that vg, jhe and ilp-1 response to immune system stimulation was independent of behavior. Younger bees were able to mount a stronger immune response than older bees, thus highlighting age as an important factor for immunity. Taken together, our results provide new insights into how age and behavior affect the honey bee's immune response.

Molecular characterization of a cDNA encoding prophenoloxidase and its expression in Apis mellifera.

Phenoloxidase (PO), a melanin-synthesizing enzyme known to play an important role in insect defense, is found as a zymogen (ProPO) in hemolymph and cuticle, where it is activated by proteolysis. We characterized the first proPO cDNA in an eusocial insect, the Apis mellifera honey bee. The AmproPO cDNA contains an ORF of 2079 bp encoding 693 amino acids, and is composed of 9 exons and 8 introns. Southern blot of digested genomic DNA suggested that only one copy of the proPO gene is present in A. mellifera. The molecular mass of the deduced ProPO and the active enzyme was predicted to be 80.1 and 74.4 kDa, respectively. The calculated pI was 6.28. BLASTp search of the deduced amino acid sequence, and neighbor-joining analysis, showed similarity with ProPOs from other insects, ranging from 47% to 63%. Protein signature analyses revealed four conserved regions, including the two copper binding sites characteristic of arthropod ProPOs. RT-PCR and Southern blot showed the highest amount of AmproPO transcripts in workers whole body, followed by queens and drones. Expression was also detected in hemocytes and integument. Real time RT-PCR showed higher amounts of AmproPO transcripts in adults and older pupae than in younger pupae and larvae, suggesting a function of AmproPO in adult exoskeleton melanization and differentiation.

Phenoloxidase activity in Apis mellifera honey bee pupae, and ecdysteroid-dependent expression of the prophenoloxidase mRNA.

Phenoloxidase (monophenol, l-dopa: oxygen oxidoreductase, EC 1.14.18.1) is a multicopper oxidase, which plays an important role in melanin synthesis, necessary for defense against intruding microorganisms and parasites, wound healing and cuticle pigmentation. A phenoloxidase from the hemolymph of honey bee pupae exhibited an apparent molecular mass of 70 kDa, as estimated by gel filtration and SDS-PAGE. Optimal pH and temperature were 6.5 and 20 degrees C, respectively. Activity was fully stable for 30 min at 50 degrees C. Like phenoloxidases from the hemolymph of other insects, the honey bee enzyme was activated by trypsin and inhibited by protease inhibitors and phenylthiourea. Only high concentrations of sodium azide effectively inhibited the detected activity. A low concentration (5 microM) of Ca2+, Mg2+, and Mn2+ had a stimulatory effect on the activity. Single Michaelis-Menten curves were observed for l-dopa and dopamine oxidation, but the affinity of the enzyme for dopamine was greater than for L-dopa. Semiquantitative RT-PCR and Southern blot analysis using a 359 bp labeled probe, and quantification of the prophenoloxidase mRNA levels by real-time PCR showed increased amounts of transcripts in hemocytes and integument from young pupae injected with 20-hydroxyecdysone.

Bacterial infection activates the immune system response and dysregulates microRNA expression in honey bees.

In insects, a rapid and massive synthesis of antimicrobial peptides (AMPs) is activated through signaling pathways (Toll and Imd) to combat invading microbial pathogens. However, it is still unclear whether different types of bacteria provoke specific responses. Immune response mechanisms and the activation of specific genes were investigated by challenging Apis mellifera workers with the Gram-negative bacterium Serratia marcescens or the Gram-positive bacterium Micrococcus luteus. The immune system responded by activating most genes of the Toll and Imd pathways, particularly AMP genes. However, genes specifically regulated by M. luteus or S. marcescens were not detected, suggesting an interaction between the signaling pathways that lead to immune effectors synthesis. Despite this finding, kappaB motifs in the 5'-UTRs of selected genes suggest a pathway-specific control of AMP and transferrin-1 gene expression. Regulation by miRNAs was also investigated and revealed a number of candidates for the post-transcriptional regulation of immune genes in bees.

Potential costs of bacterial infection on storage protein gene expression and reproduction in queenless Apis mellifera worker bees on distinct dietary regimes.

Insects are able to combat infection by initiating an efficient immune response that involves synthesizing antimicrobial peptides and a range of other defense molecules. These responses may be costly to the organism, resulting in it exploiting endogenous resources to maintain homeostasis or support defense to the detriment of other physiological needs. We used queenless worker bees on distinct dietary regimes that may alter hemolymph protein storage and ovary activation to investigate the physiological costs of infection with Serratia marcescens. The expression of the genes encoding the storage proteins vitellogenin and hexamerin 70a, the vitellogenin receptor, and vasa (which has a putative role in reproduction), was impaired in the infected bees. This impairment was mainly evident in the bees fed beebread, which caused significantly higher expression of these genes than did royal jelly or syrup, and this was confirmed at the vitellogenin and hexamerin 70a protein levels. Beebread was also the only diet that promoted ovary activation in the queenless bees, but this activation was significantly impaired by the infection. The expression of the genes encoding the storage proteins apolipophorins-I and -III and the lipophorin receptor was not altered by infection regardless the diet provided to the bees. Similarly, the storage of apolipophorin-I in the hemolymph was only slightly impaired by the infection, independently of the supplied diet. Taken together these results indicate that, infection demands a physiological cost from the transcription of specific protein storage-related genes and from the reproductive capacity.

New data on freshwater psammic Gastrotricha from Brazil.

Current knowledge of freshwater gastrotrich fauna from Brazil is underestimated as only two studies are available. The present communication is a taxonomic account of the first-ever survey of freshwater Gastrotricha in Minas Gerais State. Samplings were carried out yielding six species of three Chaetonotidae genera: Aspidiophorus cf. pleustonicus, Ichthydium cf. chaetiferum, Chaetonotus acanthocephalus, Chaetonotus heideri, Chaetonotus cf. succinctus, Chaetonotus sp., and also an undescribed species belonging to the genus Redudasys (incertae sedis): this is the first finding of specimens of Redudasys outside of original type locality. These preliminary observations suggest that the knowledge of the biodiversity of Gastrotricha in the Minas Gerais State, as well as in the whole Brazil, will certainly increase as further investigations are undertaken, and that freshwater Macrodasyida may be more common than previously thought.

Pollen diet for in vitro rearing of africanized honey bee larvae, Apis mellifera (Hymenoptera: Apidae) / Dieta polínica para criação in vitro de larvas de abelhas melíferas africanizadas, Apis mellifera (Hymenoptera: Apidae)

Pollen is the major protein source for honey bees, Apis mellifera. It is essential for the adults to produce royal jelly to feed the larvae. Young larvae receive the brood food, whereas the older (over 3 days old) larvae receive pollen in addition to brood food. The nutritional value of pollen has been investigated only in adults or at the colony level. Protocols for rearing Africanized honey bee larvae in vitro using diets with mixtures of pollen had not been established. We examined different concentrations (2.5, 5 and 10%) of two mixtures of pollen in the larval diet. The effects of pollen diets on larval development were assessed. The survival and development of larvae fed with 10% pollen was impaired; this concentration should be avoided. Concentrations of 2.5 and 5% pollen did not show significant changes in survival, weight, development or the hemolymph protein profile when compared to the controls (without pollen). However, differences in larval survival were observed between the two pollen mixtures (pollen blends 1 and 2), suggesting that a diet with a superior digestibility and greater familial diversity of pollen (blend 2) is more nutritionally adequate.

O pólen é a principal fonte de proteína para as abelhas melíferas, Apis mellifera. Ele é essencial para que os adultos produzam geleia real para nutrir as larvas. As larvas jovens recebem geleia real, enquanto que as larvas mais velhas (mais que 3 dias de idade) recebem pólen juntamente com a geleia real. O valor nutricional do pólen tem sido investigado apenas em adultos ou ao nível de colônia. Protocolos de criação de larvas de abelhas africanizadas in vitro utilizando dietas com misturas de pólen não foram estabelecidas. Nós examinamos diferentes concentrações (2,5, 5 e 10%) de duas misturas de pólen na dieta de larvas. O efeito das dietas de pólen no desenvolvimento larval foi avaliado. A sobrevivência e o desenvolvimento das larvas alimentadas com 10% de pólen foram prejudicados; esta concentração deve ser evitada. Concentrações de 2,5 e 5% de pólen não mostraram uma mudança significativa na sobrevivência, no peso, no desenvolvimento ou no perfil proteico da hemolinfa, quando comparado com os controles (sem pólen). No entanto, diferenças na sobrevivência das larvas foram observadas entre duas misturas de pólen (mistura 1 e 2), sugerindo que uma dieta com uma digestibilidade superior e maior diversidade de famílias de pólens (mistura 2) é nutricionalmente mais adequada.

Vitellogenin expression in queen ovaries and in larvae of both sexes of Apis mellifera.

In the honeybee, Apis mellifera, vitellogenin (Vg) expression has been detected in the ovary of queens, but not in that of workers. In addition, larvae of both sexes produce Vg in significant amounts, which suggest that Vg serves for functions additional to oocyte growth and energy supply to the embryo. In vivo hormone treatment experiments suggest that the decrease of 20-hydroxyecdysone concentration occurring in previtellogenic phases allows Vg production. Southern analysis indicates that the Vg gene is present as a single copy in the honeybee genome.