High-resolution (mtDNA) melting analysis for simple and efficient characterization of Africanized honey bee Apis mellifera (Hymenoptera:Apidae).

Analysis of the mtDNA variation in Apis mellifera L. has allowed distinguishing subspecies and evolutionary lineages by means of different molecular methods; from RFLP, to PCR-RFLP and direct sequencing. Likewise, geometric morphometrics (GM) has been used to distinguish Africanized honey bees with a high degree of consistency with studies using molecular information. High-resolution fusion analysis (HRM) allows one to quickly identify sequence polymorphisms by comparing DNA melting curves in short amplicons generated by real-time PCR (qPCR). The objective of this work was to implement the HRM technique in the diagnosis of Africanization of colonies of A. mellifera from Argentina, using GM as a validation method. DNA was extracted from 60 A. mellifera colonies for mitotype identification. Samples were initially analyzed by HRM, through qPCRs of two regions (485 bp/385 bp) of the mitochondrial cytochrome b gene (cytb). This technique was then optimizing to amplify a smaller PCR product (207 bp) for the HRM diagnosis for the Africanization of colonies. Of the 60 colony samples analyzed, 41 were classified as colonies of European origin whereas 19 revealed African origin. All the samples classified by HRM were correctly validated by GM, demonstrating that this technique could be implemented for a rapid identification of African mitotypes in Apis mellifera samples.

Phenotypic and genetic characterization of Africanized Apis mellifera colonies with natural tolerance to Varroa destructor and contrasting defensive behavior.

Africanized Apis mellifera colonies with promising characteristics for beekeeping have been detected in northern Argentina (subtropical climate) and are considered of interest for breeding programs. Integral evaluation of this feral material revealed high colony strength and resistance/tolerance to brood diseases. However, these Africanized honeybees (AHB) also showed variable negative behavioral traits for beekeeping, such as defensiveness, tendency to swarm and avoidance behavior. We developed a protocol for the selection of AHB stocks based on defensive behavior and characterized contrasting colonies for this trait using NGS technologies. For this purpose, population and behavioral parameters were surveyed throughout a beekeeping season in nine daughter colonies obtained from a mother colony (A1 mitochondrial haplotype) with valuable characteristics (tolerance to the mite Varroa destructor, high colony strength and low defensiveness). A Defensive Behavior Index was developed and tested in the colonies under study. Mother and two daughter colonies displaying contrasting defensive behavior were analyzed by ddRADseq. High-quality DNA samples were obtained from 16 workers of each colony. Six pooled samples, including two replicates of each of the three colonies, were processed. A total of 12,971 SNPs were detected against the reference genome of A. mellifera, 142 of which showed significant differences between colonies. We detected SNPs in coding regions, lncRNA, miRNA, rRNA, tRNA, among others. From the original data set, we also identified 647 SNPs located in protein-coding regions, 128 of which are related to 21 genes previously associated with defensive behavior, such as dop3 and dopR2, CaMKII and ADAR, obp9 and obp10, and members of the 5-HT family. We discuss the obtained results by considering the influence of polyandry and paternal lineages on the defensive behavior in AHB and provide baseline information to use this innovative molecular approach, ddRADseq, to assist in the selection and evaluation of honey bee stocks showing low defensive behavior for commercial uses.

Expression of MRJP3 and HSP70 mRNA Levels in Apis mellifera L. Workers after Dietary Supplementation with Proteins, Prebiotics, and Probiotics.

Royal jelly is an essential substance for the development of bees from larval to adult stages. Studies have identified a group of key proteins in royal jelly, denominated major royal jelly proteins (MRJPs). The group currently consists of nine proteins (MRJP1-MRJP9), with MRJP1 being the most abundant and MRJP3 being used as a microsatellite marker for the selection of queens with a greater production of royal jelly. The diet of bees is mostly composed of proteins, and supplementing this intake to encourage a higher production of their primary product is important for producers. It is estimated that, by adding probiotic and prebiotic organisms to their diets, the benefits to bees will be even greater, both for their immune systems and primary responses to stress. Circumstances that are adverse compared to those of the natural habitat of bees eventually substantially interfere with bee behavior. Stress situations are modulated by proteins termed heat shock proteins (HSPs). Among these, HSP70 has been shown to exhibit abundance changes whenever bees experience unusual situations of stress. Thus, we sought to supplement A. mellifera bee colony diets with proteins and prebiotic and probiotic components, and to evaluate the expression levels of MRJP3 and HSP70 mRNAs using qRT-PCR. The results revealed that differences in the expression of MRJP3 can be attributed to the different types of feed offered. Significant differences were evident when comparing the expression levels of MRJP3 and HSP70, suggesting that protein supplementation with pre/probiotics promotes positive results in royal jelly synthesis carried out by honey bee nurses.

Africanized Honey Bee Sting-Induced Stress-Related Cardiomyopathy: A Bee or Octopus Trap.

The Africanized honey bee commonly referred to as the "killer bee," is a hybrid of the East African lowland honey bee with various European honey bees. These bees tend to be more hostile as compared to other bee species. Their stings may have devastating clinical sequelae, including cardiovascular, cerebrovascular events, and fatalities. We report the first-in-Caribbean case of a middle-aged woman who experienced stress-related, Takotsubo cardiomyopathy (also known as stress-related cardiomyopathy [SRC]) after being afflicted with innumerable Africanized honey bee stings. Key clinical message: The clinician should be cognizant of Takotsubo's cardiomyopathy as a potential sequela of Hymenoptera envenomation and anaphylaxis.

Africanization of a feral honey bee (Apis mellifera) population in South Texas: does a decade make a difference?

The arrival to the United States of the Africanized honey bee, a hybrid between European subspecies and the African subspecies Apis mellifera scutellata, is a remarkable model for the study of biological invasions. This immigration has created an opportunity to study the dynamics of secondary contact of honey bee subspecies from African and European lineages in a feral population in South Texas. An 11-year survey of this population (1991-2001) showed that mitochondrial haplotype frequencies changed drastically over time from a resident population of eastern and western European maternal ancestry, to a population dominated by the African haplotype. A subsequent study of the nuclear genome showed that the Africanization process included bidirectional gene flow between European and Africanized honey bees, giving rise to a new panmictic mixture of A. m. scutellata- and European-derived genes. In this study, we examined gene flow patterns in the same population 23 years after the first hybridization event occurred. We found 28 active colonies inhabiting 92 tree cavities surveyed in a 5.14 km(2) area, resulting in a colony density of 5.4 colonies/km(2). Of these 28 colonies, 25 were of A. m. scutellata maternal ancestry, and three were of western European maternal ancestry. No colonies of eastern European maternal ancestry were detected, although they were present in the earlier samples. Nuclear DNA revealed little change in the introgression of A. m. scutellata-derived genes into the population compared to previous surveys. Our results suggest this feral population remains an admixed swarm with continued low levels of European ancestry and a greater presence of African-derived mitochondrial genetic composition.

Manipulation of colony environment modulates honey bee aggression and brain gene expression.

The social environment plays an essential role in shaping behavior for most animals. Social effects on behavior are often linked to changes in brain gene expression. In the honey bee (Apis mellifera L.), social modulation of individual aggression allows colonies to adjust the intensity with which they defend their hive in response to predation threat. Previous research has showed social effects on both aggression and aggression-related brain gene expression in honey bees, caused by alarm pheromone and unknown factors related to colony genotype. For example, some bees from less aggressive genetic stock reared in colonies with genetic predispositions toward increased aggression show both increased aggression and more aggressive-like brain gene expression profiles. We tested the hypothesis that exposure to a colony environment influenced by high levels of predation threat results in increased aggression and aggressive-like gene expression patterns in individual bees. We assessed gene expression using four marker genes. Experimentally induced predation threats modified behavior, but the effect was opposite of our predictions: disturbed colonies showed decreased aggression. Disturbed colonies also decreased foraging activity, suggesting that they did not habituate to threats; other explanations for this finding are discussed. Bees in disturbed colonies also showed changes in brain gene expression, some of which paralleled behavioral findings. These results show that bee aggression and associated molecular processes are subject to complex social influences.

Toxicidade de produtos fitossanitários utilizados em citros para Apis mellifera / Toxicity of pesticides used in citrus crop to Apis mellifera

O objetivo deste trabalho foi avaliar a toxicidade de inseticidas/acaricidas utilizados em cultura de citros para operárias africanizadas de Apis mellifera Linnaeus. A exposição das abelhas aos compostos foi realizada usando-se técnicas de pulverização, contaminação da dieta e contato em superfícies tratadas (folhas de citros e placas de Petri), empregando-se asdoses máximas recomendadas para a cultura. Os bioensaios foram realizados em laboratório a 25±2°C, UR 70±10% efotofase de 12h, sendo os dados de mortalidade submetidos à análise estatística, e as médias comparadas por contraste, obtendo-se grupos de efeitos semelhantes. Independente do modo de exposição, o acefato foi extremamente tóxico, matando mais de 90,0% das abelhas 24h após a aplicação. Os produtos espirodiclofeno e piriproxifem, quando aplicados diretamente sobre as abelhas, causaram níveis de mortalidade de 11,0 e 15,0%, respectivamente; os compostos buprofezina, enxofre e tetradifona apresentaram níveis de mortalidade ainda menores, com média de 5,0% entre eles. Para os ensaios decontaminação de superfície (folhas de citros e placas de Petri) e contaminação de alimento, foram obtidos dois grupos de toxicidade, um grupo somente com acefato e outro, com buprofezina, enxofre, espirodiclofeno, piriproxifem, tetradifona e água. A mortalidade média para esse segundo grupo, após 96h do início da exposição, foi de 31,0; 8,3 e 15,7%, respectivamente, para cada método de contaminação.

The aim of this research was to evaluate the toxicity of several acaricides/insecticides used in Brazilian citrus crop to africanized workers of Apis mellifera Linnaeus. The expositionof honey bees to the chemicals was performed by direct spraying, contamination of food, and contact in treated surface (citrus leaves and Petri dishes), using recommended rates ofapplication. The assays were carried out at 25±2°C, RH 70±10%, 12h of photophase and the data was statisticallyanalyzed, with mean values of mortality being compared through cluster analysis. In all assays acephate was highly toxic, with mortality at 24 hours around 90.0%. When spirodiclofen and pyriproxyfen, was sprayed directly into the honeybees, they caused mortality levels of 11.0 and 15.0%, respectively; buprofezin, sulphur and tetradifon were less toxic, with mean mortality of 5.0% among these compounds. For the assays from contamination surface (citrus leaves and Petri dishes) and food, two groups of chemicals with the same toxiceffects were observed, one with acephate and other with buprofezin, sulphur, spirodiclofen, pyriproxyfen, tetradifon and water. The average mortality after 96 hours of exposition was 31.0; 8.3 and 15.7%, respectively, for each method of contamination.

Ecological impatt on native bees by the invasive africanized honey eee / Abejas cleptoparasitas, con énfasis en las abejas hospederas colectoras de aceites (Hymenoptera: Apoidea)

Very little effort has been made to investigate bee population dynamics among intact wilderness areas. The presence of newly-arrived feral Africanized honey bee (AHB), Apis mellifera (Apidae), populations was studied for 10-17 years in areas previously with few or no escaped European apiary honey bees. Here I describe and interpret the major results from studies in three neotropical forests: French Guiana, Panama and Yucatan, Mexico (5° to 19° N. latitude). The exotic Africanized honey bees did not produce a negative effect on native bees, including species that were solitary or highly eusocial. Major differences over time were found in honey bee abundance on flowers near habitat experiencing the greatest degree of disturbance, compared to deep forest areas. At the population level, sampled at nest blocks, or at flower patches, or at light traps, there was no sudden decline in bees after AHB arrival, and relatively steady or sinusoidal population dynamics. However, the native bees shifted their foraging time or floral species. A principal conclusion is that such competition is silent, in floristically rich habitats, because bees compensate behaviorally for competition. Other factors limit their populations.

Pocos estudios han considerado la dinámica de poblaciones de abejas en bosques o hábitats no alterados por el hombre. La presencia de abejas silvestres Africanizadas de Apis mellifera (Apidae) fue estudiado por 10-17 años en áreas previamente sin esta especie. Aquí presento e interpreto resultados de tres bosques neotropicales: Guyana Francesa, Panamá y Yucatán, México (5° a 19° N. latitud). La abeja Africanizada exótica no produjo efecto negativo en las abejas nativas, incluyendo especies altamente sociales y solitarias. Diferencias mayores a través del tiempo fueron encontradas en la abundancia de las abejas de miel en flores cerca de hábitat con mayor grado de disturbio, comparado con el bosque espeso. Al nivel poblacional, muestreado en bloques de nidos trampa, en flores o con trampas ultravioletas de insectos, no hubo disminución pronta de abejas, y sí hubo una población relativamente estable o sinusoidal. Sin embargo, las abejas nativas cambiaron su hora de buscar provisiones o su selección de especies florales. Una conclusión principal es que esta competencia por los recursos es ‘silenciosa';, en las áreas florísticamente ricas estudiadas, porque las mismas abejas compensan con su comportamiento. Otros factores rigen sus poblaciones.