The effect of ambient temperature fluctuations on Culicoides biting midges population dynamics and activity in dairy farms: a longitudinal study.

The effect of climatic factors on the presence of Culicoides Latreille (Diptera: Ceratopogonidae) was previously studied. Nevertheless, lack of laboratory rearing data hampers species-specific prediction of weather fluctuations effect on population size. To determine fluctuations in population size in the field, we recorded Culicoides and other Nematocerans in seven Israeli dairy farms over two-years (2011-2012) and analysed the association of their dynamics with fluctuations in ambient temperature and total rainfall. In six farms, the most abundant species were Culicoides imicola Kieffer and Culicoides schultzei (Enderlein) gp., primarily composed of parous females, and in one farm Culicoides obsoletus (Meigen) gp., mostly nulliparous females, were dominant. While the total number of insects was similar in both years, Culicoides numbers were significantly higher in 2012, but appeared later in the season and reached a higher peak. A multi-variable linear regression model demonstrated positive association of C. imicola and C. schultzei numbers with the monthly multi-annual ambient temperature and its specific deviation, but not with monthly rainfall. C. obsoletus populations peaked at spring and sharply decreased when temperature exceeded 20 °C, and were best modelled by adding quadratic terms. Weather-specific estimation of population size under field conditions may enable to predict outbreaks intensity of Culicoides-borne viruses.

Potential tick vectors for Theileria equi in Israel.

Theileria equi Mehlhorn and Schein, 1998 (Piroplasmida: Babesiidae) is an important tick-borne pathogen of horses that is highly endemic in many parts of the world, including Israel. The present study evaluated the potential roles of five hard tick species [Hyalomma excavatum Koch, 1844; Hyalomma marginatum Koch, 1844; Rhipicephalus turanicus Pomerantsev 1936; Rhipicephalus annulatus Say, 1821; Haemaphysalis parva (Neumann, 1897) (all: Ixodida: Ixodidae)], previously found to infest horses in Israel, in acting as vectors for piroplasmosis. For this, DNA was extracted from whole ticks and, when possible, from the salivary glands in each species (n = 10-59). Polymerase chain reaction amplification and sequencing of the 18S rRNA gene were used to detect T. equi in 48 of the 127 ticks (37.8%) and in 21 of the 90 extracted salivary glands (23.3%) in all five species. All but two sequences were classified as T. equi genotype A; the remaining two were classified as genotype D. The findings of this study point to Ha. parva and R. annulatus as potential novel vectors of T. equi, and suggest that parasite genotype selection occurs within the tick vector.

Characterization and identification of microbial communities in bovine necrotic vulvovaginitis.

Bovine necrotic vulvovaginitis (BNVV) is a severe and potentially fatal disease of post-partum cows that emerged in Israel after large dairy herds were merged. While post-partum cows are commonly affected by mild vulvovaginitis (BVV), in BNVV these benign mucosal abrasions develop into progressive deep necrotic lesions leading to sepsis and death if untreated. The etiology of BNVV is still unknown and a single pathogenic agent has not been found. We hypothesized that BNVV is a polymicrobial disease where the normally benign vaginal microbiome is remodeled and affects the local immune response. To this end, we compared the histopathological changes and the microbial communities using 16S rDNA metagenetic technique in biopsies taken from vaginal lesions in post-partum cows affected by BVV and BNVV. The hallmark of BNVV was the formation of complex polymicrobial communities in the submucosal fascia and abrogation of neutrophil recruitment in these lesions. Additionally, there was a marked difference in the composition of bacterial communities in the BNVV lesions in comparison to the benign BVV lesions. This difference was characterized by the abundance of Bacteroidetes and lower total community membership in BNVV. Indicator taxa for BNVV were Parvimonas, Porphyromonas, unclassified Veillonellaceae, Mycoplasma and Bacteroidetes, whereas unclassified Clostridiales was an indicator for BVV. The results support a polymicrobial etiology for BNVV.

High relative abundance of the stable fly Stomoxys calcitrans is associated with lumpy skin disease outbreaks in Israeli dairy farms.

The vector of lumpy skin disease (LSD), a viral disease affecting Bovidae, is currently unknown. To evaluate the possible vector of LSD virus (LSDV) under field conditions, a yearlong trapping of dipterans was conducted in dairy farms that had been affected by LSD, 1-2 years previously. This was done in order to calculate monthly relative abundances of each dipteran in each farm throughout the year. The relative abundances of Stomoxys calcitrans (Diptera: Muscidae) in the months parallel to the outbreaks (December and April) were significantly higher than those of other dipterans. A stable fly population model based on weather parameters for the affected area was used to validate these findings. Its results were significantly correlated with S. calcitrans abundance. This model, based on weather parameters during the epidemic years showed that S. calcitrans populations peaked in the months of LSD onset in the studied farms. These observations and model predictions revealed a lower abundance of stable flies during October and November, when LSD affected adjacent grazing beef herds. These findings therefore suggest that S. calcitrans is a potential vector of LSD in dairy farms and that another vector is probably involved in LSDV transmission in grazing herds. These findings should be followed up with vector competence studies.

Colonization of the Mediterranean basin by the vector biting midge species Culicoides imicola: an old story.

Understanding the demographic history and genetic make-up of colonizing species is critical for inferring population sources and colonization routes. This is of main interest for designing accurate control measures in areas newly colonized by vector species of economically important pathogens. The biting midge Culicoides imicola is a major vector of orbiviruses to livestock. Historically, the distribution of this species was limited to the Afrotropical region. Entomological surveys first revealed the presence of C. imicola in the south of the Mediterranean basin by the 1970s. Following recurrent reports of massive bluetongue outbreaks since the 1990s, the presence of the species was confirmed in northern areas. In this study, we addressed the chronology and processes of C. imicola colonization in the Mediterranean basin. We characterized the genetic structure of its populations across Mediterranean and African regions using both mitochondrial and nuclear markers, and combined phylogeographical analyses with population genetics and approximate Bayesian computation. We found a west/east genetic differentiation between populations, occurring both within Africa and within the Mediterranean basin. We demonstrated that three of these groups had experienced demographic expansions in the Pleistocene, probably because of climate changes during this period. Finally, we showed that C. imicola could have colonized the Mediterranean basin in the Late Pleistocene or Early Holocene through a single event of introduction; however, we cannot exclude the hypothesis involving two routes of colonization. Thus, the recent bluetongue outbreaks are not linked to C. imicola colonization event, but rather to biological changes in the vector or the virus.

Temporal association of ambient temperature and relative humidity with Spirocerca lupi infection of Onthophagus sellatus: a 14-year longitudinal study.

The parasitic nematode Spirocerca lupi causes major morbidity and mortality in dogs. The scarab beetle Onthophagus sellatus is its major intermediate host in Israel. We investigated the prevalence of beetle infection by S. lupi in different years between 1994 and 2008. The average monthly maximum and minimum relative humidity (RH) and ambient temperature (AT) throughout the study period were calculated based on daily meteorological data. The infection prevalence decreased over the study period, possibly due to a chronological change resulting from increased preventive treatment of dogs against S. lupi, or climate change. Multivariate analysis was performed for these two hypotheses. Under the first hypothesis, chronological change was forced into the model, and environmental variables were inserted stepwise. The final model included beetle-collection date, minimum RH (RH min) during the month preceding beetle collection, its interaction with maximal AT (AT max) during that same month, and the interaction of maximal RH (RH max) and AT max, during the month of beetle collection. Under the second hypothesis, chronological change was not forced. The final model included RH max during the month of beetle collection, average RH (RHave) during the month preceding beetle collection, and its interaction with AT max during the latter month. The results suggest that under both hypotheses, RH and AT during the month preceding beetle collection influence S. lupi's ability to develop and survive in O. sellatus, and may be used to predict the risk to dogs of S. lupi infection.

Assessment of survival and body size variation of Culicoides imicola (Diptera: Ceratopogonidae) as functions of "Candidatus Cardinium" (Bacteroidetes) infection status.

"Candidatus Cardinium hertigii" (Bacteroidetes) is a maternally inherited endosymbiont known from several arthropods. Its mechanisms for persistence in host populations are mostly reproductive manipulation, though it has been occasionally reported to improve fitness parameters in several hosts. In Culicoides (Diptera: Ceratopogonidae) biting midges, the prevalence of "Candidatus Cardinium" infection was documented as moderate, with no detectable sex bias. We therefore investigated whether "Candidatus Cardinium" affects important fitness parameters, such as survival and body size, in Culicoides imicola, a dominant vector species. Field-collected midges were trapped and analyzed for survival under different environmental conditions and antibiotic treatment, taking into account "Candidatus Cardinium" infection status and parity status (i.e., parous or nulliparous). Additionally, wing lengths were measured as a proxy parameter for body size and analyzed together with "Candidatus Cardinium" infection data. The findings revealed no difference in survival of Culicoides infected with "Candidatus Cardinium" and that of uninfected midges in both parity states and under all tested conditions: optimal, starvation, heat, and antibiotic treatment. Beyond survival, no wing length difference was found for "Candidatus Cardinium"-infected versus uninfected midges. In aggregate, these findings support our conclusion that "Candidatus Cardinium" does not have an overt effect on the survival and size of adult C. imicola midges. "Candidatus Cardinium" may affect immature stages or may alter adult reproductive performance.

Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci.

The sweet potato whitefly, Bemisia tabaci, harbors Portiera aleyrodidarum, an obligatory symbiotic bacterium, as well as several secondary symbionts including Rickettsia, Hamiltonella, Wolbachia, Arsenophonus, Cardinium and Fritschea, the function of which is unknown. Bemisia tabaci is a species complex composed of numerous biotypes, which may differ from each other both genetically and biologically. Only the B and Q biotypes have been reported from Israel. Secondary symbiont infection frequencies of Israeli laboratory and field populations of B. tabaci from various host plants were determined by PCR, in order to test for correlation between bacterial composition to biotype and host plant. Hamiltonella was detected only in populations of the B biotype, while Wolbachia and Arsenophonus were found only in the Q biotype (33% and 87% infection, respectively). Rickettsia was abundant in both biotypes. Cardinium and Fritschea were not found in any of the populations. No differences in secondary symbionts were found among host plants within the B biotype; but within the Q biotype, all whiteflies collected from sage harboured both Rickettsia and Arsenophonus, an infection frequency which was significantly higher than those found in association with all other host plants. The association found between whitefly biotypes and secondary symbionts suggests a possible contribution of these bacteria to host characteristics such as insecticide resistance, host range, virus transmission and speciation.

A newly discovered bacterium associated with parthenogenesis and a change in host selection behavior in parasitoid wasps.

The symbiotic bacterium Wolbachia pipientis has been considered unique in its ability to cause multiple reproductive anomalies in its arthropod hosts. Here we report that an undescribed bacterium is vertically transmitted and associated with thelytokous parthenogenetic reproduction in Encarsia, a genus of parasitoid wasps. Although Wolbachia was found in only one of seven parthenogenetic Encarsia populations examined, the "Encarsia bacterium" (EB) was found in the other six. Among seven sexually reproducing populations screened, EB was present in one, and none harbored Wolbachia. Antibiotic treatment did not induce male production in Encarsia pergandiella but changed the oviposition behavior of females. Cured females accepted one host type at the same rate as control females but parasitized significantly fewer of the other host type. Phylogenetic analysis based on the 16S rDNA gene sequence places the EB in a unique clade within the Cytophaga-Flexibacter-Bacteroid group and shows EB is unrelated to the Proteobacteria, where Wolbachia and most other insect symbionts are found. These results imply evolution of the induction of parthenogenesis in a lineage other than Wolbachia. Importantly, these results also suggest that EB may modify the behavior of its wasp carrier in a way that enhances its transmission.

Wolbachia density and host fitness components in Muscidifurax uniraptor (Hymenoptera: pteromalidae).

Intracellular bacteria of the genus Wolbachia are found in a variety of arthropod hosts, where they cause various reproductive disorders. Attempts to study the fitness advantages and disadvantages of carrying these symbionts have yielded contradicting results. Using various doses of the antibiotic rifampicin, we were able to manipulate the density of Wolbachia in the uniparental parasitoid Muscidifurax uniraptor (Hymenoptera: Pteromalidae). The effect of different titers of the symbiont on the fecundity, reproductive rate, longevity, survival rate, and sex ratio of the host was measured. The data gathered show that following antibiotic treatments, the percentage of males rises at low doses of rifampicin and then drops again. The total sex ratio of offspring produced by treated mothers was positively correlated with the numbers of Wolbachia found in eggs laid by these females. No significant effects were detected with regard to the other studied fitness components. It is concluded that in M. uniraptor, Wolbachia are not posing any burden on the life history trait studied.

Phylogenetic analysis of parthenogenesis-inducing Wolbachia in the genus Aphytis (Hymenoptera: Aphelinidae).

Parthenogenesis-inducing intracellular bacteria of the genus Wolbachia are found in a variety of parasitoid wasp genera. The presence of Wolbachia in the uniparental Aphytis species A. lingnanensis Compere, A. diaspidis (Howard), A. chilensis Howard, and A. chrysomphali (Mercet) was tested using primers specific for the ftsZ gene. The symbiont was detected in all of these species. Wolbachia ftsZ genes that were sequenced from the four hosts show a high degree of similarity. Both the PCR with specific primers for group 'A' and phylogenetic analysis place these Wolbachia in group 'A'. The fact that the tested Aphytis species belong to different phylogenetic groups and harbour what seem to be almost identical Wolbachia, lends credence to the horizontal transmission hypothesis.

Parthenogenesis-inducing microorganisms in Aphytis (Hymenoptera: Aphelinidae).

Production of males in uniparental lines of two species in the parasitic wasp genus Aphytis was induced by rifampicin, and male sexual functioning was determined. Wolbachia-specific 16S rDNA primers were used in a PCR in order to: (1) assess correlation between thelytokous reproduction and the presence of Wolbachia; (2) detect the loss of Wolbachia DNA in uniparental A. lingnanensis following antibiotic treatments, with or without the presence of a host; and (3) clone and sequence part of the Wolbachia 16S rDNA from the uniparental Aphytis species for phylogenetic studies. Males produced viable sperm that was transferred to the female spermatheca following mating. However, sperm failure to effect egg fertilization resulted in all-male progeny. Wolbachia were found in the two uniparental (A. lingnanensis and A. diaspidis) but not in the two biparental (A. lingnanensis and A. melinus) Aphytis lines tested. They can be detected in wasps up to 7 days following antibiotic treatments, regardless of the presence of host. The 16S rDNA for the symbionts in the two Aphytis species is virtually identical, and is most closely related to the Wolbachia found in Muscidifurax uniraptor (Pteromalidae).

A bumetanide-sensitive, potassium carrier-mediated transport system in excitable tissues.

The binding of [3H]-bumetanide to rat brain synaptosomes revealed the existence of two binding sites. The high affinity site (R1 = 46.6 fmoles/mg protein) binds bumetanide and furosemide with Kd1 of 13 nM and 1.5 microM respectively, while the low affinity site (R2 = 1.37 nmoles/mg protein) is characterized by Kd2 of 200 microM and 680 microM for bumetanide and furosemide, respectively. Bumetanide sensitive 86Rb uptake was 34 +/- 14.5, 38.3 +/- 1.4, 18.6 +/- 1.3 and 29.0 +/- 6.1% of total 86Rb uptake in synaptic plasma membrane vesicles, rat brain synaptosomes, Neuroblastoma N1E115 cell line and chick chest muscle cells, respectively. Furosemide and bumetanide inhibited 86Rb uptake to rat brain SPM- vesicles in a dose dependent fashion. Half maximal inhibition (IC50) was observed at 20 nM and 4 microM for bumetanide and furosemide, respectively. Bumetanide-sensitive transport was dependent on extravesicular sodium and chloride concentrations with a Km of 21 and 25 mM for the two ions, respectively. These results demonstrate the existence of a "loop diuretic" sensitive carrier-mediated K+ transport system in brain and other excitable cells.

Single unit activity in the auditory cortex of a monkey performing a short term memory task.

Short term memory to tones (STMT) was investigated by recording single unit activity in the auditory cortex of a behaving monkey. The activity of each unit was studied in two behavioral conditions: a) During task performance, the monkey had to compare two tones separated by one second of silence (inter-stimulus interval). b) During a non-performing period; the monkey heard the two tones but did not respond behaviorally. It was noted that the firing rate of many units during the inter-stimulus interval (ISI) was dependent on the frequency of the first tone. Such dependency was observed even towards the end of the ISI, both during task performance trials (50% of the units) and during the non-performing period (32% of the units). The activity of these units could be the basis of STMT in both of these behavioral states. In 65% of all the units tested, the responses during the ISI were of a higher magnitude in the performance period than were the responses in the non-performance period. The activity of these units may be related either to general processes such as attention and expectation or to short-term memory processes. During task performance, the responses of 23% of the units to the second tone were dependent on whether its frequency was identical to that of the first tone. Such dependency was never observed during the non-performing period. These units may detect similarity or non similarity between two tones presented one second apart. Periodic patterns of firing were not found in the study, thus suggesting that the ISI responses were not generated by reverberatory activity in simple closed loops. On the basis of these results, several alternative mechanisms of STMT are suggested.

The effects of stimuli on the activity and functional connectivity of local neuronal groups in the cat auditory cortex.

Simultaneous extracellular recordings from one electrode of 'local' groups of 3-6 neurons were obtained from the auditory cortex of unanesthetized, paralyzed cats. The activity and functional connectivity of local microenvironments were examined under various auditory stimuli. Single cell response patterns were examined using peri-stimulus (PST) histograms and functional connectivity among neighboring cells by the cross renewal density (CRD) histograms. Analysis of the PST histograms suggested that a high percentage of single cells demonstrated different response patterns to different stimuli. Analysis of the CRD histograms suggested, on the one hand, that only small numbers of neighboring cells behaved as if there were direct connections from one cell to another, and that these direct connections appeared to be excitatory. On the other hand, many cell pairs shared input from shared sources which lay outside the local groups. The majority of functional connections were altered by at least one of the stimuli delivered, thus demonstrating the system's plasticity. It is suggested that long-term gates at the synaptic level are responsible for this phenomenon.