Regulation of vole populations by the nematode <Emphasis Type="Italic">Trichuris arvicolae</Emphasis>: insights from modelling

The goal of this study is to determine whether a parasitic nematode may regulate, or destabilise by inducing demographic cycles, its host populations. We explore three host–parasite systems through population dynamic models. The hosts considered are the fossorial water vole, Arvicola terrestris, the common vole Microtus arvalis and the bank vole Myodes (Clethrionomys) glareolus and the parasitic nematode is Trichuris arvicolae. Three differential equation-based mathematical models are developed including host immunity and the existence of trade-off between immunity and host survival. Using parameters estimated from field data and laboratory observations, all these models show that T. arvicolae can induce host population regulation but not demographic cycles. The regulation effect of the nematode is un-ambiguous for the water vole (reduction of 50.2% of the host population size), but less obvious for the common vole (5.9%) and even less for the bank vole (1.4%). Important biological parameters to be taken into account in such models are discussed. Experimental confirmation of the regulatory potential of the nematode and of the costs of mounting an immune response against this nematode are now required. Communicated by W. Lutz     

Microsatellite markers for the common vole (<Emphasis Type="Italic">Microtus arvalis</Emphasis>) and their cross-species utility

Fragmentation of natural habitats of the common vole (Microtus arvalis) provides an excellent model system to study the consequences of restricted gene flow and small population sizes for isolated populations. Here we describe the isolation and characteristics of 10 autosomal and one X-linked microsatellite marker. These new markers were tested in 24 voles from a natural population in eastern Germany. Loci were highly polymorphic with numbers of alleles per locus ranging from three to 26 and expected heterozygosities from 0.51 to 0.97. All loci except for the X-linked locus Mar105 followed Hardy–Weinberg expectations. Cross-species amplifications revealed that most loci were polymorphic as well in M. agrestis, M. thomasi, and M. pennsylvanicus.     

Fasting in the American marten (<Emphasis Type="Italic">Martes americana</Emphasis>): a physiological model of the adaptations of a lean-bodied animal

The American marten (Martes americana) is a boreal forest marten with low body adiposity throughout the year. The aim of this study was to investigate the adaptations of this lean-bodied species to fasting for an ecologically relevant duration (48 h) by exposing eight farm-bred animals to total food deprivation with seven control animals. Selected morphological and hematological parameters, plasma and serum biochemistry, endocrinological variables and liver and white adipose tissue (WAT) enzyme activities were determined. After 48 h without food, the marten were within phase II of fasting with depleted liver and muscle glycogen stores, but with active lipid mobilization indicated by the high lipase activities in several WAT depots. The plasma ghrelin concentrations were higher due to food deprivation, possibly increasing appetite and enhancing foraging behavior. The lower plasma insulin and higher cortisol concentrations could mediate augmented lipolysis and the lower triiodothyronine levels could suppress the metabolic rate. Fasting did not affect the plasma levels of stress-associated catecholamines or variables indicating tissue damage. In general, the adaptations to short-term fasting exhibited some differences compared to the related farm-bred American mink (Mustela vison), an example of which was the better ability of the marten to hydrolyze lipids despite its significantly lower initial fat mass.     

Acaricidal effects of <Emphasis Type="Italic">Corymbia citriodora</Emphasis> oil containing <Emphasis Type="Italic">para</Emphasis>-menthane-3,8-diol against nymphs of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (Acari: Ixodidae)

The toxicity of para-menthane-3,8-diol (PMD), the main arthropod-repellent compound in the oil of the lemon eucalyptus, Corymbia citriodora, was evaluated against nymphs of Ixodes ricinus using five methods (A–E) of a contact toxicity bioassay. Mortality rates were estimated by recording numbers of dead nymphs at 30 min intervals during the first 5 h after the start of exposure and at longer intervals thereafter. The mortality rate increased with increasing concentration of PMD and duration of exposure with a distinct effect after 3.5 h. From the results obtained by methods A, C and E, the LC₅₀ range was 0.035–0.037 mg PMD/cm² and the LC₉₅ range was 0.095–0.097 mg PMD/cm² at 4 h of exposure; the LT₅₀ range was 2.1–2.8 h and the LT₉₅ range was 3.9–4.2 h at 0.1 mg PMD/cm². To determine the duration of toxic activity of PMD, different concentrations (0.002, 0.01, 0.1 mg PMD/cm²) were tested and mortality was recorded at each concentration after 1 h; thereafter new ticks were tested. This test revealed that the lethal activity of PMD remained for 24 h but appeared absent after 48 h. The overall results show that PMD is toxic to nymphs of I. ricinus and may be useful for tick control.     

Adaptations to fasting in the American mink (<Emphasis Type="Italic">Mustela vison</Emphasis>): nitrogen metabolism

The aim of this study was to investigate the adaptations of protein metabolism to seasonal fasting in an actively wintering boreal carnivore. Fifty farm-bred male American minks Mustela vison were divided into a fed control group and four experimental groups fasted for 2, 3, 5 or 7 days. The responses of nitrogen metabolism to wintertime food deprivation were determined by measuring the rate of weight loss, the tissue total protein concentrations and the plasma amino acid, urea, ammonia, uric acid and total protein levels. The mink has relatively poor adaptations to food deprivation, as it is not able to prolong phase II of fasting with fat as the major metabolic fuel. Instead, the species has to derive a part of its energy requirements from the breakdown of body proteins. The end product of protein catabolism—urea— accumulates in its circulation, and the mink may not be able to recycle urea-N. Although the mink can still have a high body fat percent at the end of the 7-day fast, it appears to enter phase III of fasting with stimulated proteolysis during this period.     

An efficient process for production and purification of hyaluronic acid from <Emphasis Type="Italic">Streptococcus </Emphasis><Emphasis Type="Italic">equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Growth of Streptococcus zooepidemicus in a 10 l bioreactor with 50 g sucrose/l and 10 g casein hydrolysate/l gave 5–6 g hyaluronic acid/l after 24–28 h. Purification of hyaluronic acid gave a recovery of 65% with the final material having an Mr of ∼4 × 10⁶ Da with less than 0.1% protein.     

Time Course of <Emphasis Type="Italic">c-fos</Emphasis>, <Emphasis Type="Italic">vasopressin</Emphasis> and <Emphasis Type="Italic">oxytocin</Emphasis> mRNA Expression in the Hypothalamus Following Long-Term Dehydration

Summary 1. This study presents a time course analysis of the messenger RNA (mRNA) levels of c-fos, vasopressin (VP), and oxytocin (OT) in the paraventricular (PVN) and supraoptic nucleus (SON), following acute and chronic dehydration by water deprivation. 2. Male Wistar rats were separated into five groups: nondehydrated (control group) and dehydrated for 6, 24, 48 and 72 h. Following water deprivation, animals were decapitated, their blood was collected for hematocrit, osmolality, and plasma sodium measurements, and brains were removed for dissection of both PVN and SON. 3. As expected, the hematocrit, osmolality, plasma sodium, and weight loss were increased after water deprivation. In SON, a significant increase in both VP and OT mRNA expression was observed 6 h after dehydration reaching a peak at 24 h and returning to basal levels of expression at 72 h. In the PVN, an increase in both VP and OT mRNA expression occurred 24 h after dehydration. At 72 h the VP and OT mRNA expression levels had decreased but they were still at higher levels than those detected in control animals. 4. These results suggest that SON is the first nucleus to respond to the dehydration stimulus. Additionally, we also observed an increase in c-fos mRNA expression in both PVN and SON 6 h after water deprivation, which progressively decreased 24, 48, and 72 h after the onset of water deprivation. Therefore, it is possible that c-fos may be involved in the modulation of VP and OT genes, regulating the mRNA expression levels on a temporally distinct basis within the PVN and SON.     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

Induction of pathogenesis-related proteins during biocontrol of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> with <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.) plants

To investigate the biocontrol effectiveness of the antibiotic producing bacterium, Pseudomonas aureofaciens 63–28 against the phytopathogen Rhizoctonia solani AG-4 on Petri plates and in soybean roots, growth response and induction of PR-proteins were estimated after inoculation with P. aureofaciens 63–28 (P), with R. solani AG-4 (R), or with P. aureofaciens 63–28 + R. solani AG-4 (P + R). P. aureofaciens 63–28 showed strong antifungal activity against R. solani AG-4 pathogens in Petri plates. Treatment with P. aureofaciens 63–28 alone increased the emergence rate, shoot fresh weight, shoot dry weight and root fresh weight at 7 days after inoculation, when compared to R. solani AG-4; P + R treatment showed similar effects. Peroxidase (POD) and β-1,3-glucanase activity of P. aureofaciens 63–28 treated roots increased by 41.1 and 49.9%, respectively, compared to control roots. POD was 26% greater in P + R treated roots than R. solani treated roots. Two POD isozymes (59 and 27 kDa) were strongly induced in P + R treated roots. The apparent molecular weight of chitinase from treated roots, as determined through SDS-PAGE separation and comparison with standards, was about 29 kDa. Five β-1,3-glucanase isozymes (80, 70, 50, 46 and 19 kDa) were observed in all treatments. These results suggest that inoculation of soybean plants with P. aureofaciens 63–28 elevates plant growth inhibition by R. solani AG-4 and activates PR-proteins, potentially through induction of systemic resistance mechanisms.     

Feeding rate of sibling vole <Emphasis Type="Italic">Microtus rossiaemeridionalis</Emphasis> and mandarin vole <Emphasis Type="Italic">Lasiopodomys mandarinus</Emphasis> housed in cages and enclosures

Consumption of food of different nutritive value by sibling vole Microtus rossiaemeridionalis and mandarin vole Lasiopodomys mandarinus as a function of housing conditions—in cages or enclosures—was comparatively studied. Experimental groups included 8–14 adult non-reproducing animals of both sexes kept one per cage or enclosure. The rate of food consumption and digestibility were studied by standard methods and the obtained data were statistically analyzed using Statistica 6.0. Significant differences in the rate of food consumption by mandarin voles housed under different conditions as well as in the consumption and digestibility of food of different biochemical composition in both vole species have been revealed.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Pelecyphora aselliformis</Emphasis> erhenberg (cactaceae)

Summary In vitro propagation of Pelecyphora aselliformis, a Mexican cactus which is considered rare and is highly valued in the commercial market, was initiated using seeds as explants. The longitudinal explants from seedlings germinated in vitro were cultivated on Murashige and Skoog medium containing 8.8 μM benzyladenine (BA) or 4.6 μM kinetin at pH 7.0. After 120 d, each explant gave rise to five shoots and this number of shoots increased 20–25% after subculture. The hyperhydricity was similar in both media, but callus formation was lower on the medium with BA. The shoot development, in terms of epicotyl length, and fresh and dry weight after 6 wk, was also recorded. The epicotyl length was similar on shoot-forming media but the quality of shoots was better on media containing BA. In about 1 yr, 500–600 well-defined shoots were obtained. The rooting of shoots was very slow and a vigorous radical system was observed after 1 yr of culture.     

Familiarity and partner preferences in female common voles, <Emphasis Type="Italic">Microtus arvalis</Emphasis>

Captive female common voles (Microtus arvalis) had a clear social preference for familiar males in a standard preference test. Cohabitation for a short period resulted in preferences for familiar partners, and females spent eight times more time in body contact with a familiar male than with an unfamiliar male. Females also displayed stranger-directed aggression. Our results suggest that the common vole is a species with selective partner preferences and a tendency to form tight social relationships and stable male–female associations.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

Local coexistence and niche differences between the Lusitanian and Mediterranean pine voles (<Emphasis Type="Italic">Microtus lusitanicus</Emphasis> and <Emphasis Type="Italic">M. duodecimcostatus</Emphasis>)

In the present study, we analyzed the coexistence pattern of the Lusitanian pine vole (Microtus lusitanicus) and the Mediterranean pine vole (Microtus duodecimcostatus) in a potential area of sympatry in a Mediterranean landscape (Portugal). We also determined the relative contribution of local, landscape, and spatial factors explaining the differences in the distribution patterns of the two species in the region. Using a kriging interpolation method, we obtained a map of sympatric and allopatric areas of species occurrence. The estimated sympatry area corresponded to a northwest–southeast belt representing 11.3% of the study area. Habitat niche differences were assessed with binomial GLMs followed by a variance partitioning. At a local scale, higher altitude, higher cover of shrubs, lower clay content in the soil, and lower cover of tree canopy were the most important factors distinguishing M. lusitanicus presence sites from those with M. duodecimcostatus. At a larger scale, the presence of forest landscape units and the low abundance of “montado” units were the most influencing landscape factors in the identification of M. lusitanicus occurrence sites when compared to M. duodecimcostatus. Our results suggested that local coexistence of M. lusitanicus and M. duodecimcostatus in the field is a rare event. The differences in distribution patterns of the two pine vole species were mostly explained by fine-scale environmental factors and by shared spatial effects.     

Expression of early developmental genes in vole <Emphasis Type="Italic">Microtus rossiaemeridionalis</Emphasis>

The expression of genes Sox2, Klf4, Myc, Sall4, Gata6, Foxa2, Hnf4a, Cdx2, Esrrb, Hand1 in cell lines, embryos and organs of adult voles Microtus rossiaemeridionalis was studied. High resemblance of the expression patterns of these genes in the organs of adult voles, mice and humans was demonstrated. It was established that genes Gata6, Foxa2 and Hnf4a were specifically expressed in vole extraembryonic endoderm cells, while Cdx2 and Hand1 genes, in trophoblast stem cells. This shows that these genes can be used as markers of corresponding vole cell lines. Indirect confirmation pointing to the fact that Oct4 gene is a marker gene for epiblast cells both in the vole and mouse was obtained.     

Submerged culture of a mycelial formulation of a bioherbicidal strain of <Emphasis Type="Italic">Myrothecium </Emphasis><Emphasis Type="Italic">verrucaria</Emphasis> with mitigated mycotoxin production

A mycelial formulation of the fungus Myrothecium verrucaria (IMI 361690) containing 0.20% Silwet L-77 surfactant was found to be highly efficacious in controlling the exotic invasive weed kudzu. The mycelium can be rapidly (48–72 h) produced in several media, including an inexpensive soy flour–corn meal medium. Mycelial yields were 2, 10, and 25 g dry weight l⁻¹ in Czapek-Dox, Richard’s V-8, and soy flour–corn meal media, respectively. Scale-up production in soy flour–corn meal medium using laboratory fermenters (10–25 l), resulted in a mycelial formulation that caused 90% mortality of naturally-occurring mature (0.9–1.0 m in height) kudzu within 48 h after application in field experiments. HPLC analyses revealed that the mycelium produced in this liquid culture contained no detectable amounts of the trichothecene mycotoxins roridin A and verrucarin A (limit of detection 2 μg ml⁻¹). This has resulted in a safer, yet effective bioherbicidal product. We anticipate that these findings should improve the probability of EPA registration and subsequent commercial development of this bioherbicide.     

Persistence of <Emphasis Type="Italic">Paranosema</Emphasis> (<Emphasis Type="Italic">Nosema</Emphasis>) <Emphasis Type="Italic">locustae</Emphasis> (Microsporidia: Nosematidae) among grasshopper (Orthoptera: Acrididae) populations in the Inner Mongolia Rangeland,China

Paranosema (Nosema) locustae Canning has persisted in grasshopper populations in the Inner Mongolia Rangeland since spore treatment was applied in 1993. In the treatment year, a 58.32–71.43% reduction of grasshoppers was recorded and 59.26–80.36% of surviving grasshoppers of various species were infected. In the years following application, prevalence of P. locustae varied from 13.65% to 60.71%, demonstrating that P. locustae remains in grasshopper populations for many years. Infection prevalence was more than 60.00% for Dasyhippus barbipes (F.-W.), Bryodema luctuosum luctuosoma (Stoll) and Angaracris barabensis (Pall.) in 1993 but was very variable, ranging from 20.00% to 60.71% over the subsequent nine years. Disease prevalence was 43.72–60.71% in 1993 and 1994 for Oedaleus asiaticus B.-Bienko and Myrmeleotettix palpalis (Zub.) but gradually declined to approximately 20%, where it remained. Persistence of P. locustae in grasshopper populations may be important in keeping densities below economic thresholds for a number of years after application. Handling Editor: Helen Roy.     

Effects produced by the feeding of larvae of <Emphasis Type="Italic">Ornithodoros</Emphasis> aff. <Emphasis Type="Italic">puertoricensis</Emphasis> (Acari: Argasidae) on laboratory mice

The clinical picture produced by the feeding of larvae of Ornithodoros aff. puertoricensis on laboratory mice, was studied using different larval infestation protocols that included 30, 40 or 50 larvae per mouse and control uninfested groups. Clinical effects appeared around 72 h of larval feeding, having a first stage characterized by hyperaemia in both nasal and ocular mucosa, followed by respiratory symptoms (96–120 h) and nervous incoordination (120–144 h). No one mouse evidenced paralysis, and nervous symptoms were never observed in animals infested with only 30 larvae. High mortality (commonly up to 70%) was observed in mice with respiratory symptoms, while 100% of animals in the nervous phase died between 168 and 192 h after the beginning of larval feeding. When some infested mice were treated with a solution of Amitraz the larvae were killed and reversion of symptoms was observed. These effects are ascribed to the presence of a toxin in the saliva of the feeding larvae.