P. De la Rúa

Estimating the Density of Honeybee Colonies across Their Natural Range to Fill the Gap in Pollinator Decline Censuses

Although pollinator declines are a global biodiversity threat, the demography of the western honeybee (Apis mellifera) has not been considered by conservationists because it is biased by the activity of beekeepers. To fill this gap in pollinator decline censuses and to provide a broad picture of the current status of honeybees across their natural range, we used microsatellite genetic markers to estimate colony densities and genetic diversity at different locations in Europe, Africa, and central Asia that had different patterns of land use. Genetic diversity and colony densities were highest in South Africa and lowest in Northern Europe and were correlated with mean annual temperature. Confounding factors not related to climate, however, are also likely to influence genetic diversity and colony densities in honeybee populations. Land use showed a significantly negative influence over genetic diversity and the density of honeybee colonies over all sampling locations. In Europe honeybees sampled in nature reserves had genetic diversity and colony densities similar to those sampled in agricultural landscapes, which suggests that the former are not wild but may have come from managed hives. Other results also support this idea: putative wild bees were rare in our European samples, and the mean estimated density of honeybee colonies on the continent closely resembled the reported mean number of managed hives. Current densities of European honeybee populations are in the same range as those found in the adverse climatic conditions of the Kalahari and Saharan deserts, which suggests that beekeeping activities do not compensate for the loss of wild colonies. Our findings highlight the importance of reconsidering the conservation status of honeybees in Europe and of regarding beekeeping not only as a profitable business for producing honey, but also as an essential component of biodiversity conservation.

Genetic structure and distinctness of Apis mellifera L. populations from the Canary Islands

The genetic structure of Apis mellifera populations from the Canary Islands has been assessed by mitochondrial (restriction fragment length polymorphisms of the intergenic transfer RNAleu‐COII region) and nuclear (microsatellites) studies. These populations show a low level of genetic variation in terms of average number of alleles and degree of heterozygosity. Significant differences in the distribution of alleles were found in both data sets, confirming the genetic differentiation among some of the islands but not within them. Two mitochondrial haplotypes characteristic of the Canary Islands are found at high frequencies, although populations are introgressed by imported honeybees of eastern European C lineage. This introgression is rather high on Tenerife and El Hierro and low on Gran Canaria and La Gomera, whereas on La Palma it has not been recorded. The finding of microsatellite alleles characteristic of the eastern European lineage corroborates the genetic introgression. Phylogenetic analyses indicate that the Canarian honeybees are differentiated from other lineages and provide genetic evidence of their African origin.

Mitochondrial DNA variability in the Canary Islands honeybees (Apis mellifera L.).

The mitochondrial DNA (mtDNA) of individuals from 79 colonies of Apis mellifera from five Canary Islands was studied using the DraI test based on the restriction of PCR products of the tRNAleu–COII intergenic region. Five haplotypes of the African (A) lineage and one of the west European (C) lineage were found. The haplotypes A14 and A15 are described for the first time. These haplotypes have a new P sequence named P1. The wide distribution and high frequency of haplotype A15 suggest that it is characteristic of the Canarian Archipelago. Sources of haplotype variability of honeybee mtDNA in the Canary Islands (waves of colonization from Africa, queen importations, habitat diversification) are discussed.

Distribution patterns of the Q and B biotypes of Bemisia tabaci in the mediterranean basin based on microsatellite variation

At least five of the biotypes described in the Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) complex are known to be present in the Mediterranean Basin area. Only two of them, however, are economically relevant, that is, biotypes B and Q. Biological and genetic differences between the two biotypes have been well studied, but less is known about their patterns of genetic variation and population structure. To address these issues, a study was undertaken based on variation at six microsatellite loci among a subset of nine B. tabaci populations (five belonging to the Q and four to the B biotype). The data obtained show that (i) these loci showed considerable polymorphism in the Q and B biotypes populations although the presence of null alleles can obscure the picture; (ii) the Iberian‐Q, Canarian‐Q, and Egyptian‐B populations exhibit heterozygosity excess as a result of bottleneck events; (iii) the low genetic differentiation between the Israeli, Iberian Peninsula, and Italian populations suggest that these populations share a common gene pool; (iv) the genetic distances between the Canarian‐Q population and the geographically close population from Morocco indicates spatial isolation and a limited gene flow; and finally (v) the microsatellite data for the B populations indicate that the whiteflies from Egypt and Israel have a close phylogenetic relationship, but the source of these biotype B invasions into the Mediterranean area remains unknown.

Evidence for widespread Leishmania infantum infection among wild carnivores in L. infantum periendemic northern Spain

Leishmania spp. infection was investigated in tissue samples of wild carnivores from the Spanish Basque Country (BC), by PCR and DNA sequencing. The region is at the northern periphery of Leishmania infantum endemic Iberian Peninsula and infection in the dog (reservoir) or other species has not been previously reported. Leishmania kinetoplast DNA was detected by real-time PCR (rtPCR) in 28% (44/156) of animals. Specifically, in 26% of Eurasian badgers (n=53), 29% of foxes (n=48), 29% of stone martens (n=21) and in 25-50% of less numerous species including genets, wild cats, pole cats, European mink and weasels. Infected animals particularly badgers, were most prevalent in the southernmost province of the BC (Araba) in areas dominated by arable land. Subsequent amplification and sequencing of a fragment of the rRNA internal transcribed spacer 2 (ITS2) from a subset of rtPCR positives samples confirmed the species as L. infantum, showing a high sequence homogeneity with ITS2 sequences of L. infantum from dogs and humans from southern Spain. In summary, this study reports for the first time L. infantum infection in wild carnivores from the BC including in stone martens, pole cats and minks in which infection has not been previously described. It supports the need to study infection in dogs and people in this region and is an example of the value of infection surveillance in wildlife to assess potential risks in the domestic environment and their role in spreading infections in non-endemic areas.

Spatial distribution of human asymptomatic Leishmania infantum infection in southeast Spain: A study of environmental, demographic and social risk factors

Recent PCR studies indicate that asymptomatic L. infantum infection is common in people in southern Europe. Understanding its spatial distribution is a requisite to evaluate the public health implications and to design disease control schemes. We investigated infection in blood samples from 657 donors in southeast Spain using PCR and antibody ELISA. They came from 19 blood centers and were interviewed about their residence, occupation, dog ownership and Leishmaniosis awareness. The percentage of PCR and ELISA positives were 8% (49/618) and 2% (13/657). Donors residences were spatially clustered around blood donning centers and PCR prevalence was 18% in rural municipalities with 20-1330 inhabitants, 12% in those with 1467-5088 inhabitants and 3% in larger communities, and was associated with dog ownership (p<0.05). Further analysis of data from rural donors indicated that PCR status was strongly related to the climate, altitude and soil type in the donors residence area and not to other demographic or sociologic variables. Mixed logistic regression analysis predicted PCR prevalence to be greatest in the 200-300m altitude range with a mean spring-summer (time of highest vector activity) temperature of 18.4-19.0°C. A temperature and altitude risk map was generated that will provide the basis for elaborating evidence-based vector surveillance studies.

Assessment of the mitochondrial origin of honey bees from Argentina

Summary The molecular diversity of the honey bee Apis mellifera in the province of Buenos Aires (Argentina) has been analysed through the study of the mitochondrial DNA. The mitochondrial haplotype corresponding to the intergenic region tRNAleu-COII has been determined in samples from 300 colonies from 150 apiaries distributed in 71 localities of the province of Buenos Aires. Eight different haplotypes have been found, four of the African, three of the West European and one of the East European evolutionary lineages. The frequency of these haplotypes corroborates that the European yellow honey bee Apis mellifera ligustica is predominant in the Buenos Aires province whereas the black European bee A. m. mellifera is rare. The presence of the African haplotypes A1 and A4 common in Africanized honey bees, confirms a border of the africanization process located around the 30°-35° SL parallels. The other two African haplotypes (A8 and A11) are indicative of an Iberian or North African origin of some colonies. The influence of the transhumance and beekeeping practices is discussed in relation to the genetic variability detected.

Novel Cytochrome P450 Genes, CYP6EB1 and CYP6EC1, are Over-Expressed in Acrinathrin-Resistant Frankliniella occidentalis (Thysanoptera: Thripidae)

ABSTRACT Control of Frankliniella occidentalis (Pergande) is a serious problem for agriculture all over the world because of the limited range of insecticides that are available. Insecticide resistance in F. occidentalis has been reported for all major insecticide groups. Our previous studies showed that cytochrome P450-mediated detoxification is a major mechanism responsible for insecticide resistance in this pest. Degenerate polymerase chain reaction was used to identify P450 genes that might be involved in acrinathrin resistance, in a laboratory population of F. occidentalis. Associated sequences were classified as belonging to the CYP4 and CYP6 families. Real-time quantitative polymerase chain reaction analyses revealed that two genes, CYP6EB1 and CYP6EC1, were over-expressed in adults and L2 larvae of the resistant population, when compared with the susceptible population, suggesting their possible involvement in resistance to acrinathrin.

Sequence and RFLP analysis of the ITS2 ribosomal DNA in two Neotropical social bees, Melipona beecheii and Melipona yucatanica (Apidae, Meliponini)

Abstract.Two stingless bees species of the genus Melipona, M. beecheii and M. yucatanica, are the only ones reported for the Yucatan Peninsula. The natural distribution of M. beecheii ranges from southern Mexico to Costa Rica, that of M. yucatanica from south Mexico to Guatemala. Colonies of both species occur in a variety of habitats and show adaptations to local conditions denoting the occurrence of ecotypes. The ITS2 of ribosomal DNA has been characterized in both species and its utility to discriminate among colonies has been investigated through RFLP experiments. The ITS2 region is unusually long, 1788 bp in M. beecheii and 1845 bp in M. yucatanica (including the 3′ end of the 5.8S gene and partial 5′ of the 28S gene). Mean nucleotide divergence between both ITS2 sequences is 16% (excluding sites with insertions/deletions) and 20% when the insertions/deletions are taken into account. The G+C content in both sequences is close to 53%. The PCR-RFLP assay was performed with 12 restriction enzymes on colonies of M. beecheii from Mexico (Yucatan, Campeche and Chiapas) Costa Rica, El Salvador and Guatemala, and of M. yucatanica from Mexico (Yucatan) and Guatemala. The restriction patterns obtained allow to discriminating colonies of both species with different origins. Both kinds of data are thus useful for assessing intra and interspecific genetic variability and for developing appropriate conservation strategies for these species.

Morphometric affinities and population structure of honey bees of the Balearic Islands (Spain)

SUMMARY Morphometric analyses of the honey bees (Apis mellifera) of the Balearic Islands revealed two statistically separable morphoclusters: (1) Menorca and Mallorca and (2) Ibiza and Formentera. These morphoclusters are geographically juxtaposed between North African and Iberian morphoclusters. The Mahalanobis distances between centroids of the four morphoclusters demonstrate that the two Balearic morphoclusters have closer affinities to the North African bees, than to southern Iberian and lastly northern Iberian bees. The morphometric data closely complement that of mitochondrial DNA that also shows that Balearic bees derive from African lineage.