EDUARDO CESARIn Apis mellifera, the bee that produces commercial honey, tasks are well distributed among the colonyEDUARDO CESAR
Someone who wanders onto the campus of the University of São Paulo (USP) in Ribeirão Preto and is attracted by the beauty of a building flanked by four reflecting pools and a coral vine bush at the entrance may be in for a surprise. Next to this beautiful garden there is a lawn with a hundred wooden boxes supported on small pedestals, like altars. Those who are curious and want to get a closer look will realize that they have wandered into the midst of an open-air apiary. It is there, literally in the middle of a colony of native stingless bees, that biologists Zilá Simões, Klaus Hartfelder and Márcia Bitondi are trying to understand how the social structure and behavior of these insects are encoded in their DNA. The colonies of Apis mellifera (bees with stingers commonly used in honey production) are in a separate area, where there is no risk of any unsuspecting visitor entering alone.
The three researchers are at the heart of the Bee Development Biology Laboratory (LBDA), a network of researchers in various São Paulo institutions (see Pesquisa FAPESP Issue nº 130). The group participated in the consortium that mapped the Apis mellifera genome and more recently the mandaçaia bee, the Melipona quadrifasciata, and they are now preparing the sequencing of another Brazilian species, the marmalade-yellow (Frieseomelitta varia). And this does not include the dozens of studies on specific functions of bee development genes.
In the decade that followed the publication of the first bee genome, in an article in the journal Nature in 2006, the group began to benefit from the same set of tools that evolved from the work on the human genome and changed the biomedical research landscape. In addition to obtaining the complete DNA sequence of the insects, the researchers now have the tools and know-how available to generate and analyze transcriptomes. The term is a reference to the analysis of genes that are being transcribed, that is, active in the production of proteins in various tissues of bees.
When analyzing the results, the researchers were able to see the network of interaction between the transcribed genes, such as which genes influence other gene expressions and which are connected to other aspects of interest. In a recent study, for example, the group was able to identify the role of genes involved in the development of worker bees. They have a specific structure on their rear legs to carry pollen, the corbicula or pollen basket, which is absent in queen bees. As each larva can develop into a queen or worker bee, according to the food it receives, only the transcriptomes will reveal how they differ. Using genomic and bioinformatic techniques, the Ribeirão Preto scientists were able to identify genes of the Hox type, which control most of the body’s development and are important in the formation of the corbicula; they described the discovery in a 2012 article in the journal PLOS One. One of these genes, the Ubx, had a level of expression 25 times higher during the pupal stage of the worker bees, when compared to that of the queen bees, and revealed an essential key to their caste differentiation.
JULIANA RAMOS MARTINSNourishing chambers feed oocyte in ovariole of the queen…JULIANA RAMOS MARTINS
Hox genes belong to a large family of genes often targeted by developmental biology researchers to determine which structures of the body are derived from each segment of an embryo. In some cases, however, it is necessary to look at more subtle parts of the genome to understand how the functions are distributed in a colony. In a subsequent study published in the same journal in 2013, Hartfelder and his collaborators described some regulatory sequences, known as long non-coding RNAs, which influence the size of bee ovaries. These molecules, along with microRNAs, are key components of the caste differentiation process, by causing, for example, the destruction of many ovarian cells of the workers. As a result, the reproductive system of the queens is much larger, a characteristic fundamental to constant egg production.
Transcriptomes
In order to do this kind of study, the LBDA and its collaborators have already produced over 100 transcriptomes of different tissues of the insect, at different times of life. The challenge is to understand in detail how caste development is programmed into the DNA and is triggered by diet—notably, queen bees are fed more royal jelly than worker bees at key moments of their development.
Understanding bee development has always been considered a challenge. The organization of these insects contradicts a principle of biology, namely that evolution is unable to create long-lived animals that also have a high rate of reproduction. It is necessary to forego one thing in favor of another. The queen bee, however, typically lives more than a year (a long time for an insect) and has a high rate of reproduction, able to lay up to half a million eggs throughout her life. This is possible thanks to the division of labor with worker bees, who live approximately one month without reproducing.
This remarkable division of labor within the colony was the target of an international study in which LBDA participated—the main reason for the Ribeirão Preto laboratory’s analysis of the DNA of the mandaçaia bee. There is a spectrum of socialization when it comes to all species of bees. Some have a more pronounced caste division (eusocial), while most females live a solitary existence. The group compared the genomes of 10 different types of bees and found that the more eusocial and hierarchical a species is, the more regulatory sequences of genes there are in the DNA, as shown in an article published in June 2015 in the journal Science. In the case of Apis mellifera, when the queen dies, the workers produce another queen, by feeding a larva with royal jelly. In less eusocial species, the workers compete with the queen for reproduction, while in the non-social species all females reproduce. In the mandaçaia, a highly social native bee, the division of labor is very clear, but the workers participating in the colony’s reproduction produce males.
The network of interaction between these stretches of DNA—which do not contain their own protein recipes, but influence the activity of other stretches—is so prolific that Simões is poised to create a new term to define it: reguloma. “This word is not yet used, but the practical study of this has already begun,” she says.
JULIANA RAMOS MARTINS… and muscles (in red) cover intestine of worker. In blue, cell nuclei JULIANA RAMOS MARTINS
Bioinformatics
Having mastered the genomic tools, the LBDA researchers have generated so much information that work can no longer be done in the same way as in the past. What the most advanced bee biology laboratories are doing today is essentially what the American geneticist Eric Lander (one of the fathers of the Human Genome Project) called “doing science without an hypothesis,” which upsets many biologists. With genomics, scientists can look at the molecular functioning of an organism without an initial concept of which gene does what, by selecting the genes to be studied through algorithms that analyze the networks of interaction between them.
It is unclear whether the genomics methodology is fundamentally different from other areas of science, but certainly the way geneticists work has changed a lot. “One of the key pieces today is to have good bioinformaticians; we can not live without them anymore,” says Bitondi. “And one laboratory cannot do it alone. We need to bring together the expertise of several laboratories.”
At USP in Ribeirão Preto, the demand for this type of researcher was filled through graduate projects with the Genetics Department providing part of the training, but the computer science knowledge had to be sought elsewhere. Such was the career path of Daniel Guariz Pinheiro, who graduated from USP’s Institute of Mathematics and Statistics (IME) and did postdoctoral training at the Ribeirão Preto Genetics Department. Today he is a professor at São Paulo State University (Unesp), Jaboticabal campus, and the principal bioinformatician of LBDA’s collaboration network.
In addition to working on transcriptomes, one of the tasks of the bioinformatician is to organize the genomes on which the studies are based, because the sequencing machines obtain DNA fragments that need to be lined up correctly. “Bioinformatics then comes into play by assembling the pieces of this puzzle, connecting parts in order to obtain the complete genome sequence,” says Guariz, who contributed to the paper published in Science. “We are already more than 99% there, especially in the case of Apis mellifera.”
One of the prominent works to which the LBDA scientists contributed was an international collaboration led by the University of Uppsala, Sweden, for which the genomes of 140 honey bees from around the world, and belonging to different populations, were sequenced. The genetic diversity map of Apis mellifera, published in 2014 in the journal Nature Genetics, suggests a different origin for the species. It was believed that it would have emerged from Africa, but the study points to its spreading from Asia, where today the other bees of the genus Apis also live.
Intervention
The Ribeirão Preto biologists do not just work and live on the computer, however. One of their core activities is to perform experiments to prove the hypotheses raised by genomics. Using a technique known as RNA interference, the LBDA researchers can turn off the expression of specific genes in bees to study their functionality.
This kind of basic research also has practical implications, given the marked reductions in populations of several species of bees in which viruses and pesticides are identified as the main culprits. In addition to the ecological impact of these factors, which are still being studied, agricultural losses of fruits, grains and other plants that depend on bees for pollination must also be considered (see sidebar on p. 50). Two species whose genomes have recently been sequenced and analyzed with the participation of LBDA are bees of the genus Bombus, common name bumblebee, and the Brazilian carpenter bees, of importance for environmental pollination services. The findings of the study were published in June in the journal Genome Biology. The researchers found that the Bombus, whose sociality is much less complex, have many genes that were believed to be exclusive to Apis. The pattern of RNA expression was markedly different between these two genera, however, reinforcing the idea that the key to understanding the behavior of these insects lies in the regulome.
To find the best way to deal with the reductions in bee populations, the researchers say, genomic research may provide assistance in three areas: comparing bee species, colonies of the same species, and individuals of the same colony (the latter through the study of transcriptomes). The Ribeirão Preto group has developed the expertise to work on all of these aspects.
LÉO RAMOSScaptotrigona on açaí flowers, in Pará State, northern BrazilLÉO RAMOS
Pollination by bees supports $12 billion worth of Brazilian agriculture
For at least two decades, hives in various regions of the world have been experiencing reduced populations. The phenomenon, which was given the name Colony Collapse Disorder (or CCD), has been well documented in Europe and North America, where populations have dwindled up to 50%, but it has been little studied in Brazil (see Pesquisa FAPESP Issue No. 137). A recent study led by biologist Tereza Cristina Giannini of USP concluded that the risk to Brazilian agriculture of heavy losses due to the lack of insects for pollination is very real.
Giannini and her colleagues from USP and the Federal University of Ceará (UFC) analyzed 141 plants important to Brazilian agriculture and found that 85 of them depended to some extent on pollination by bees. The study, published in the May 2015 issue of the Journal of Economic Entomology, estimates that revenues from crops that depend on pollinators would fall by 30% ($12 billion) if these insects vanished from the scene. Half of this amount represents soybean crop losses. Coffee, tomatoes, cotton, cocoa and oranges could also be greatly affected.
Even among plants that are not completely dependent on cross-pollination, the pollen transfer between different plants affects the quality of the fruit. “Studies here in Brazil have consistently demonstrated this in strawberries,” says Giannini. “The best-formed fruits are pollinated by bees. When pollination does not occur, the pulp fails to grow properly and compromises the formation of the strawberry.”
With the exception of honey production, Brazilian farmers still do not have much awareness of the importance of these insects. The notion that the presence of bees is an important environmental service is common in agriculture, yet even so, few invest in maintaining hives for pollinating crops. “The pollination service provided by bees, however, is far more valuable than the known products of the hive,” says Giannini.
In the northern hemisphere, several causes have been identified for CCD. The main ones are the use of insecticides, the emergence of pathogens, habitat loss and fragmentation, climate change, inadequate management and competition from alien species. In Brazil, however, it is not yet known which of these factors represents the greatest threat.
Many of the cultures studied by Giannini’s group have Apis mellifera as pollinators, but also include stingless bees, solitary bees of the genus Centris and carpenter bees and bumblebees (Xylocopa and Bombus). Giannini believes there is an “urgent need” for research on the reproductive biology of plants and insects to understand the extent of the problem in Brazil.
Project
Causal analysis of Apis mellifera development – regulatory genes and hierarchical networks of gene expression in the specification of tissues and organs (nº 2011/03171-5); Grant Mechanism Thematic Project; Principal Investigator Zilá Luz Paulino Simões (FFCLRP-USP); Investment R$1,029,830.00 (FAPESP).
Scientific articles
WALLBERG, A. et. al. A worldwide survey of genome sequence variation provides insight into the evolutionary history of the honeybee Apis mellifera. Nature Genetics. V. 46, No. 10, p. 1081-8, October 2014
SADD, B.M. et. al. The genomes of two key bumblebee species with primitive eusocial organization. Genome Biology. V. 16, No. 76, April 2015.
GIANNINI, T.C. et al. The dependence of crops for pollinators and the economic value of pollination in Brazil. Journal of Economic Entomology. V. 108, No. 3, p. 1-9. June 1, 2015.
KAPHEIM, K.M. et al. Genomic signatures of evolutionary transitions from solitary to group living. Science. V. 348, No. 6239, p. 1139-43. June 5, 2015.
