Tiago Mauricio Francoy

Identification of Africanized honey bees through wing morphometrics: two fast and efficient procedures*

Currently available morphometric and genetic techniques that can accurately identify Africanized honey bees are both costly and time consuming. We tested two new morphometric techniques (ABIS — Automatic Bee Identification System and geometric morphometrics analysis) on samples consisting of digital images of five worker forewings per colony. These were collected from 394 colonies of Africanized bees from all over Brazil and from colonies of African bees, Apis mellifera scutellata (n = 14), and European bees, A. m. ligustica (n = 10), A. m. mellifera (n = 15), and A. m. carnica (n=15) from the Ruttner collection in Oberursel, Germany (preserved specimens). Both methods required less than five minutes per sample, giving more than 99% correct identifications. There was just one misidentification (based on geometric morphometrics analysis) of Africanized bees compared with European subspecies, which would be the principal concern in newly-colonized areas, such as the southern USA. These new techniques are inexpensive, fast and precise.ZusammenfassungDie Afrikanisierten Honigbienen sind unter den verschiedenen Unterarten und Rassengruppen der Honigbiene (Apis mellifera L.) in den Neotropen und den Nachbarregionen am meisten respektiert und gefürchtet, insbesondere da sie in neue Gebiete einwandern. Die Identifizierung der Afrikanisierten Bienen ist in diesen Regionen für die Bewirtschaftung der Bienenvölker daher unverzichtbar. Sie ermöglicht die Bestimmung ihres Verbreitungsgebiets und ihrer Ausbreitungsgeschwindigkeit, dies ist sowohl für die Imker als auch für die damit befassten Regierungseinrichtungen von Bedeutung.Wir benutzten zwei kürzlich entwickelte morphometrische Techniken (ABIS — Automatic Bee Identification System und die Geometrische Morphometrische Analyse), um Proben aus jeweils fünf rechten Vörderflügeln pro Volk zu analysieren (Tab. I). Beide dieser Methoden benötigten in einem Vergleich von 394 über ganz Brasilien verteilten Völkern weniger als 5 Minuten pro Volk und erreichten eine mehr als 99% korrekte Identifizierung. Diese ergaben 14 Völker von A. m. scutellata, 10 Völker von A. m. ligustica, 15 Völker von A. m. mellifera und 15 Völker von A. m. carnica (Tab. II und III). Mit ABIS können einzelne Bienen bestimmt werden, während die Geometrische Morphometrische Analyse eine auf jeweils 5 Flügeln beruhende Identifikationen auf Kolonieebene durchführt. Die meisten der Fehleinordnungen fanden zwischen Afrikanisierten und Afrikanischen Bienen sowie zwischen den europäischen Unterarten statt. Nur eines der Afrikanisierten Bienenvölker wurde irrtümlich als eine europäische Unterart eingeordnet, dies ist die Fehlerart die insbesondere innerhalb von neubesiedelten Gebieten wie den Südstaaten der USA von Bedeutung wäre. Die erreichten Fortschritte in Computertechnologie, statistischen Analysen und Bilderkennungssoftware sowie die verbesserten Informationen über die relevanten Messgrößenbereiche und die höhere Genauigkeit und größere Geschwindigkeit der Messungen selbst machen es nun möglich, Afrikanisierte Bienen ausschließlich anhand von Digitalaufnahmen der Vörderflügel in Minutenschnelle zu identifizieren.

Nosema ceranae has been present in Brazil for more than three decades infecting Africanized honey bees.

Until the mid-1990s, the only microsporidium known to infect bees of the genus Apis was Nosema apis. A second species, Nosema ceranae, was first identified in 1996 from Asian honey bees; it is postulated that this parasite was transmitted from the Asian honey bee, Apis cerana, to the European honey bee, Apis mellifera. Currently, N. ceranae is found on all continents and has often been associated with honey bee colony collapse and other reports of high bee losses. Samples of Africanized drones collected in 1979, preserved in alcohol, were analyzed by light microscopy to count spores and were subjected to DNA extraction, after which duplex PCR was conducted. All molecular analyses (triplicate) indicated that the drones were infected with both N. ceranae and N. apis. PCR products were sequenced and matched to sequences reported in the GenBank (Acc. Nos. JQ639316.1 and JQ639301.1). The venation pattern of the wings of these males was compared to those of the current population living in the same area and with the pattern of drones collected in 1968 from Ribeirão Preto, SP, Brazil, from a location close to where African swarms first escaped in 1956. The morphometric results indicated that the population collected in 1979 was significantly different from the current living population, confirming its antiquity. Considering that the use of molecular tools for identifying Nosema species is relatively recent, it is possible that previous reports of infections (which used only light microscopy, without ultrastructural analysis) wrongly identified N. ceranae as N. apis. Although we can conclude that N. ceranae has been affecting Africanized honeybees in Brazil for at least 34 years, the impact of this pathogen remains unclear.

Geometric morphometrics of the wing as a tool for assigning genetic lineages and geographic origin to Melipona beecheii (Hymenoptera: Meliponini)

The stingless bee Melipona beecheii presents great variability and is considered a complex of species. In order to better understand this species complex, we need to evaluate its diversity and develop methods that allow geographic traceability of the populations. Here we present a fast, efficient, and inexpensive means to accomplish this using geometric morphometrics of wings. We collected samples from Mexico, Guatemala, El Salvador, Nicaragua, and Costa Rica and we were able to correctly assign 87.1% of the colonies to their sampling sites and 92.4% to their haplotype. We propose that geometric morphometrics of the wing could be used as a first step analysis leaving the more expensive molecular analysis only to doubtful cases.

The queen is dead—long live the workers: intraspecific parasitism by workers in the stingless bee Melipona scutellaris

Insect societies are well known for their high degree of cooperation, but their colonies can potentially be exploited by reproductive workers who lay unfertilized, male eggs, rather than work for the good of the colony. Recently, it has also been discovered that workers in bumblebees and Asian honeybees can succeed in entering and parasitizing unrelated colonies to produce their own male offspring. The aim of this study was to investigate whether such intraspecific worker parasitism might also occur in stingless bees, another group of highly social bees. Based on a large‐scale genetic study of the species Melipona scutellaris, and the genotyping of nearly 600 males from 45 colonies, we show that ∼20% of all males are workers’ sons, but that around 80% of these had genotypes that were incompatible with them being the sons of workers of the resident queen. By tracking colonies over multiple generations, we show that these males were not produced by drifted workers, but rather by workers that were the offspring of a previous, superseded queen. This means that uniquely, workers reproductively parasitize the next‐generation workforce. Our results are surprising given that most colonies were sampled many months after the previous queen had died and that workers normally only have a life expectancy of ∼30 days. It also implies that reproductive workers greatly outlive all other workers. We explain our results in the context of kin selection theory, and the fact that it pays workers more from exploiting the colony if costs are carried by less related individuals.

Intraspecific queen parasitism in a highly eusocial bee

Insect societies are well-known for their advanced cooperation, but their colonies are also vulnerable to reproductive parasitism. Here, we present a novel example of an intraspecific social parasitism in a highly eusocial bee, the stingless bee Melipona scutellaris. In particular, we provide genetic evidence which shows that, upon loss of the mother queen, many colonies are invaded by unrelated queens that fly in from unrelated hives nearby. The reasons for the occurrence of this surprising form of social parasitism may be linked to the fact that unlike honeybees, Melipona bees produce new queens in great excess of colony needs, and that this exerts much greater selection on queens to seek alternative reproductive options, such as by taking over other nests. Overall, our results are the first to demonstrate that queens in highly eusocial bees can found colonies not only via supersedure or swarming, but also by infiltrating and taking over other unrelated nests.

Integrated landmark and outline-based morphometric methods efficiently distinguish species of Euglossa (Hymenoptera, Apidae, Euglossini)

Morphometric methods permit identification of insect species and are an aid for taxonomy. Quantitative wing traits were used to identify male euglossine bees. Landmark- and outline-based methods have been primarily used independently. Here, we combine the two methods using five Euglossa. Landmark-based methods correctly classified 84% and outline-based 77%, but an integrated analysis correctly classified 91% of samples. Some species presented significantly high reclassification percentages when only wing cell contour was considered, and correct identification of specimens with damaged wings was also obtained using this methodology.

Monogamy in large bee societies: a stingless paradox

High genetic diversity is important for the functioning of large insect societies. Across the social Hymenoptera (ants, bees, and wasps), species with the largest colonies tend to have a high colony-level genetic diversity resulting from multiple queens (polygyny) or queens that mate with multiple males (polyandry). Here we studied the genetic structure of Trigona spinipes, a stingless bee species with colonies an order of magnitude larger than those of polyandrous honeybees. Genotypes of adult workers and pupae from 43 nests distributed across three Brazilian biomes showed that T. spinipes colonies are usually headed by one singly mated queen. Apart from revealing a notable exception from the general incidence of high genetic diversity in large insect societies, our results reinforce previous findings suggesting the absence of polyandry in stingless bees and provide evidence against the sperm limitation hypothesis for the evolution of polyandry. Stingless bee species with large colonies, such as T. spinipes, thus seem promising study models to unravel alternative mechanisms to increase genetic diversity within colonies or understand the adaptive value of low genetic diversity in large insect societies.

Non-destructive imaging to describe a new species of Obama land planarian (Platyhelminthes, Tricladida)

The land planarians in the genus Obama include the largest species of the Neotropical Geoplaninae. Morphological discrimination of Obama species can be difficult, as many species are morphologically similar and some present asymmetric – difficult to interpret – copulatory organs. New techniques are thus welcomed to provide faster species description and identification. Here, we study several specimens of Obama by means of mainly 2D and 3D μCT‐based images obtained through X‐ray microcomputed tomography (μCT) of a paratype, and, complementarily, histological sections of the holotype and a second paratype, which were digitized into virtual slides. Comparison of these images with traditional histological sections and descriptions of the known species of the genus allows us to conclude that our specimens constitute a new species, which we describe here. We further evaluate the phylogenetic placement of the new species using gene fragments from the mitochondrial cytochrome c oxidase subunit I and the nuclear elongation factor‐1α. Raw and derivative μCT data and virtual histological sections were deposited in an open repository (GigaDB) and are freely available. This work leads us to conclude that μCT constitutes a relatively fast, inexpensive non‐destructive method that produces results comparable to those of traditional histology, and is thus amenable for describing flatworm species.

Evaluation of inexpensive pollen substitute diets through quantification of haemolymph proteins

Depto. de Ciencia e Tecnologia, Instituto de Ciencia e Tecnologia, Universidade Federal de Sao Paulo (Unifesp), 12231-280 Sao Jose dos Campos, SP, Brazil. Received 10 October 2011, accepted subject to revision 11 October 2012, accepted for publication 14 January 2013. *Corresponding author: Email: michelle.manfrini@unifesp.br

Evaluating classification and feature selection techniques for honeybee subspecies identification using wing images

We have compared the performance of 7 classifiers to bee species identification.The use of feature selection improves the performance of classification in this domain.The best classifier to Apis mellifera subspecies identification is the Naive Bayes.We have outlined the importance of Apis mellifera to agriculture. The main pollinator commercially available, i.e. Apis mellifera, is now facing a severe population decrease worldwide due to the so-called Colony Collapse Disorder. Measures to preserve this species are urgent. Honeybees inhabit several different environments, from swamps to deserts, from high mountains to the African savannah. They are classified into several different subspecies, each one adapted to a particular set of environmental characteristics. The identification of subspecies is based on morphometric features from the entire bee body, but in the last years features from the fore wings have proven to be very efficient for classification. Several methods have been developed to perform the automatic classification through images of bee wings, and geometric morphometrics has been reported to achieve good results in terms of consumed time and reliability of the results. However, there has been no study evaluating the impact of feature selection and new classification methods on the identification performance. We here evaluate seven combinations of feature selectors and classifiers by their hit ratio with real bee wing images. Feature selection proved to be beneficial to all the evaluated combinations and the Naive Bayes classifier combined with a correlation-based feature selector achieved the best results. These conclusions can benefit researches that rely on classification by geometric morphometrics features, both for bees and for other animal species.