Effect of Temperature on the Biotic Potential of Honeybee Microsporidia

The biological cycle of Nosema spp. in honeybees depends on temperature. When expressed as total spore counts per day after infection, the biotic potentials of Nosema apis and N. ceranae at 33°C were similar, but a higher proportion of immature stages of N. ceranae than of N. apis were seen. At 25 and 37°C, the biotic potential of N. ceranae was higher than that of N. apis. The better adaptation of N. ceranae to complete its endogenous cycle at different temperatures clearly supports the observation of the different epidemiological patterns.Biotic potential represents the maximum reproductive capacity of a population under optimum environmental conditions. Thus, a species fulfilling its biotic potential would exhibit maximal exponential population growth, thereby augmenting the possibilities of transmission of the species. A wide range of factors affects the biotic potential of each species, and among the external factors, temperature clearly influences the life cycles of most parasitic species (4).Nosemosis is a common worldwide disease of adult honeybees (Apis mellifera) that is caused by microsporidia (19). Nosema apis was the only agent known to produce this disease in A. mellifera until N. ceranae was identified in this host in 2005, in Europe (11) and Taiwan (12). Both these microsporidia infect and multiply in the ventricular cells of A. mellifera, and they can be found under different environmental conditions in both the northern and southern hemispheres (17, 13). Significantly, N. ceranae seems to be more pathogenic than N. apis in caged worker bees (10, 18), and it has recently been related to significant losses of bees and colony collapses under field conditions (17, 8).Due to the lack of comparative studies of the factors affecting parasite virulence, trials were designed to determine the influence of temperature on the biotic potentials of both microsporidia. Only the deleterious effect of high exogenous temperature on spores of N. apis has been checked previously (16).In this work, purified N. apis and N. ceranae spores with a minimum viability of 99% (tested with 4% trypan blue) were obtained from experimentally infected honeybees always maintained at 33°C as described previously (9). The spores were counted using a hemocytometer chamber (19), while the Nosema species identification was confirmed by PCR (17).The experimental infection of bees was carried out as described previously (10). Briefly, young Nosema-free honeybees were starved for 2 h and fed 2 μl of 50% sucrose solution containing 100,000 viable N. ceranae or N. apis spores. Honeybees were anesthetized with CO₂, and later, a droplet of the spore solution was administered to each honeybee by touching a micropipette to its mouthparts until the entire droplet was consumed. The bees that did not consume the entire droplet were discarded. Uninfected control bees were fed 2 μl of 50% sucrose solution alone.Three trials were carried out for 1 week each at three different temperatures (25, 33, and 37°C). Each trial included four replicate cages of 30 N. apis-infected honeybees, four replicate cages of 30 N. ceranae-infected honeybees, and four replicate cages of 30 uninfected honeybees. Three different refrigerated incubators (Memmert) were used for N. ceranae- or N. apis-infected bees and controls.On days 1, 2, 3, 4, and 7 postinfection (p.i.), five living honeybees were removed from each of three cages (n = 15) in each incubator. Spore detection and counting were performed with each bee. To obtain the Nosema spore counts, the whole abdomen from each bee was placed into a sterile Eppendorf microtube in 200 μl of double-distilled water (PCR grade). After thorough grinding of the abdomen, the spore count for each bee was calculated with a hemocytometer. In addition, PCR was used to confirm the Nosema species identification. The biotic index was calculated as the total N. apis or N. ceranae spore count per day after infection.Finally, on the same days p.i., a histopathological study was performed with two honeybees from the fourth cage in each incubator. The ventriculus of each bee, with the Malpighian tubules attached, was processed as described previously (10).The daily spore count per bee and its relationship to the temperature were established with a nonparametric Mann-Whitney test for each Nosema species (level of significance, α = 0.05). Two curvilinear regression models for each temperature were established. The growth curves fitted predict the increases of the N. ceranae and N. apis spore counts over time (days p.i.), and the slopes and intercepts of the regression lines could be compared. SPSS (version 15.0) software was used to perform the tests and regression analysis.Throughout the study, Nosema infection was not evident in any of the control bees (Table ​(Table1),1), while N. ceranae was detected in all the bees (100%) exposed to this microsporidium. The proportion of the bees exposed to N. apis in which N. apis infection was detected varied during the period studied (0 to 100%).

TABLE 1.

Percentages of infected honey bees in the different test groups^a

Analysis of UV-induced mutation spectra in Escherichia coli by DNA polymerase eta from Arabidopsis thaliana

DNA polymerase eta belongs to the Y-family of DNA polymerases, enzymes that are able to synthesize past template lesions that block replication fork progression. This polymerase accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and therefore may contributes to resistance against sunlight in vivo, both ameliorating survival and decreasing the level of mutagenesis. We cloned and sequenced a cDNA from Arabidopsis thaliana which encodes a protein containing several sequence motifs characteristics of Pol eta homologues, including a highly conserved sequence reported to be present in the active site of the Y-family DNA polymerases. The gene, named AtPOLH, contains 14 exons and 13 introns and is expressed in different plant tissues. A strain from Saccharomyces cerevisiae, deficient in Pol eta activity, was transformed with a yeast expression plasmid containing the AtPOLH cDNA. The rate of survival to UV irradiation in the transformed mutant increased to similar values of the wild type yeast strain, showing that AtPOLH encodes a functional protein. In addition, when AtPOLH is expressed in Escherichia coli, a change in the mutational spectra is detected when bacteria are irradiated with UV light. This observation might indicate that AtPOLH could compete with DNA polymerase V and then bypass cyclobutane pyrimidine dimers incorporating two adenylates.     

Host specificity in spinturnicid mites: do parasites share a long evolutionary history with their host?

Host specificity in parasites can be explained by spatial isolation from other potential hosts or by specialization and speciation of specific parasite species. The first assertion is based on allopatric speciation, the latter on differential lifetime reproductive success on different available hosts. We investigated the host specificity and cophylogenetic histories of four sympatric European bat species of the genus Myotis and their ectoparasitic wing mites of the genus Spinturnix. We sampled >40 parasite specimens from each bat species and reconstructed their phylogenetic COI trees to assess host specificity. To test for cospeciation, we compared host and parasite trees for congruencies in tree topologies. Corresponding divergence events in host and parasite trees were dated using the molecular clock approach. We found two species of wing mites to be host specific and one species to occur on two unrelated hosts. Host specificity cannot be explained by isolation of host species, because we found individual parasites on other species than their native hosts. Furthermore, we found no evidence for cospeciation, but for one host switch and one sorting event. Host‐specific wing mites were several million years younger than their hosts. Speciation of hosts did not cause speciation in their respective parasites, but we found that diversification of recent host lineages coincided with a lineage split in some parasites.     

MALDI-TOF mass spectrometry as a tool for differentiation of Bradyrhizobium species: Application to the identification of Lupinus nodulating strains

Genus Bradyrhizobium includes slow growing bacteria able to nodulate different legumes as well as species isolated from plant tumours. The slow growth presented by the members of this genus and the phylogenetic closeness of most of its species difficults their identification. In the present work we applied for the first time Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to the analysis of Bradyrhizobium species after the extension of MALDI Biotyper 2.0 database with the currently valid species of this genus. With this methodology it was possible to identify strains belonging to phylogenetically closely related species of genus Bradyrhizobium allowing the discrimination among species with rrs gene identities higher than 99%. The application of MALDI-TOF MS to strains isolated from nodules of different Lupinus species in diverse geographical locations allowed their correct identification when comparing with the results of rrs gene and ITS analyses. The nodulation of Lupinus gredensis, an endemic species of the west of Spain, by B. canariense supports the European origin of this species.     

Microsporidia infecting Apis mellifera: coexistence or competition. Is Nosema ceranae replacing Nosema apis?

Nosema ceranae has been suggested to be replacing Nosema apis in some populations of Apis mellifera honeybees. However, this replacement from one to the other is not supported when studying the distribution and prevalence of both microsporidia in professional apiaries in Spanish territories (transverse study), their seasonal pattern in experimental hives with co-infection or their prevalence at individual level (either in worker bees or drones). Nevertheless, N.ceranae has shown to present a higher prevalence at all the studied levels that could indicate any advantage for its development over N.apis or that it is more adapted to Spanish conditions. Also, both microsporidia show a different pattern of preference for its development according to the prevalence in the different Spanish bioclimatic belts studied. Finally, the fact that all analyses were carried out using an Internal PCR Control (IPC) newly developed guarantees the confidence of the data extracted from the PCR analyses. This IPC provides a useful tool for laboratory detection of honeybee pathogens.     

Cost-effectiveness of habitat-suitability maps using low-detailed data for elusive bat species

European bat species are strictly protected by law, and the Member States of the European Union are obliged to record species condition and to contribute to their conservation. Habitat-suitability models are an essential aid in assessing the conservation status and distribution of a species. However, model performance depends on the data quality. This study compares habitat-suitability models that were generated from two data sets that differ in the degree of details included. The first model used data that were low in detail but freely available and the second used data that were very detailed but costly. Three hypotheses were addressed: (1) that the model using low-detailed data is sufficient in its performance to aid the assessment of species distribution and infrastructural planning; (2) the visualisation of actual species distribution is more accurate in the high-detailed model; and (3) habitat-suitability maps can depict species distribution better than species occurrence data alone. To develop models, climate, geographic and roosting data of Myotis bechsteinii were used. Models allowed very good spatial predictions of suitable habitats. However, the model using low-detailed data overestimated suitable habitat. The high-detailed model was more able to predict actual species distribution. These findings were supported by field evaluation where M. bechsteinii could only be detected in areas where both models predicted high habitat suitability. This framework is promising as it resulted in spatially explicit habitat-suitability maps and suggests that similar models may be used to improve the understanding of bat distribution and factors endangering other species of bats.     

The effects of reproductive state on digestive efficiency in three sympatric bat species of the same guild

The functional link between food as an energy source and metabolizable energy is the digestive tract. The digestive organs may change in size, structure, or retention time in response to energetic demands of the animal. Very efficient digestive tracts may be better at processing food but require higher energetic investments for maintenance even when post-absorptive. These costs influence the resting metabolic rate (RMR) that is defined as the energy necessary to fuel vital metabolic functions in a resting animal. In bats a trade-off between the necessity for a highly efficient digestive tract and moderate energetic maintenance costs may be particularly important. We hypothesized that low RMR coincides with low digestive efficiency (defined as apparent metabolizable energy coefficient (MEC)) and that phases of increased energetic demand are compensated for by increased digestive efficiency. We measured RMR and apparent MEC in the bats species Myotis nattereri, M. bechsteinii, and Plecotus auritus. In support of our hypothesis, M. nattereri has the lowest mass-specific RMR of the three species and the lowest apparent MEC. However, apparent MEC did not change during phases with differing energetic demands in any of the bat species, probably because bats operate at the limit of their sustainable energy demand.     

Azaperone and sudden death of drive net-captured southern chamois

The use of tranquilizers in the capture of southern chamois (Rupicapra pyrenaica) for scientific and/or management purposes (collection of samples, marking, translocations) was studied to improve animal welfare during capture operations. We used clinical findings and a statistical approach to analyze the causes of six incidences of mortality during captures using drive nets and tranquilizers in this species. Hematology and serum biochemistry, pathology, the use of tranquilizers and their dosages, the number of people involved in the capture of the chamois, and the location were all taken into account. The selection of candidate models to explain mortality was conducted using the theoretic information approach. Both observational findings and the models selected suggested that high doses of azaperone and to a lesser extent haloperidol had an effect on mortality rates. The higher mean serum lactate concentrations found in the chamois that died suggests that fatigue levels increased drug sensitivity and provoked the appearance of adverse effects, thereby increasing the probability of death. We conclude that butyrophenones—and especially azaperone—have a low safety margin in the southern chamois, contrary to what has been described for other species.     

Silvicolous on a Small Scale: Possibilities and Limitations of Habitat Suitability Models for Small,Elusive Mammals in Conservation Management and Landscape Planning

Species distribution and endangerment can be assessed by habitat-suitability modelling. This study addresses methodical aspects of habitat suitability modelling and includes an application example in actual species conservation and landscape planning. Models using species presence-absence data are preferable to presence-only models. In contrast to species presence data, absences are rarely recorded. Therefore, many studies generate pseudo-absence data for modelling. However, in this study model quality was higher with null samples collected in the field. Next to species data the choice of landscape data is crucial for suitability modelling. Landscape data with high resolution and ecological relevance for the study species improve model reliability and quality for small elusive mammals like Muscardinus avellanarius. For large scale assessment of species distribution, models with low-detailed data are sufficient. For regional site-specific conservation issues like a conflict-free site for new wind turbines, high-detailed regional models are needed. Even though the overlap with optimally suitable habitat for M. avellanarius was low, the installation of wind plants can pose a threat due to habitat loss and fragmentation. To conclude, modellers should clearly state the purpose of their models and choose the according level of detail for species and environmental data.