Fox Feces Research Articles

FMRI fingerprint of unconditioned fear-like behavior in rats exposed to trimethylthiazoline

Unconditioned fear plays an important yet poorly understood role in anxiety disorders, and only few neuroimaging studies have focused on evaluating the underlying neuronal mechanisms. In rodents the predator odor trimethylthiazoline (TMT), a synthetic component of fox feces, is commonly used to induce states of unconditioned fear. In this study, arterial spin labeling-based functional magnetic resonance imaging (fMRI) was applied to detect TMT-induced regional modulations of neuronal activity in Wistar rats. During TMT exposure the rats displayed increased freezing behavior and reduced exploration in the odor-associated area. Neuronal activity was selectively increased in the dorsal periaqueductal gray, superior colliculus and medial thalamus and reduced in the median raphe, locus coeruleus, nucleus accumbens shell, ventral tegmental area, ventral pallidum and entorhinal piriform cortex. This fMRI fingerprint involving distinct neuronal pathways was used to describe a schematic model of fear processing. Key brain areas known to underlie fear and anxiety-related autonomic and behavioral responses as well as centers of motivational processing were identified as being part of this functional circuitry of innate fear. Thus, preclinical fMRI studies based on unconditioned fear methods may provide a valuable translational approach to better characterize etiological and pathological processes underlying anxiety disorders.

A Fear-Inducing Odor Alters PER2 and c-Fos Expression in Brain Regions Involved in Fear Memory

Evidence demonstrates that rodents learn to associate a foot shock with time of day, indicating the formation of a fear related time-stamp memory, even in the absence of a functioning SCN. In addition, mice acquire and retain fear memory better during the early day compared to the early night. This type of memory may be regulated by circadian pacemakers outside of the SCN. As a first step in testing the hypothesis that clock genes are involved in the formation of a time-stamp fear memory, we exposed one group of mice to fox feces derived odor (TMT) at ZT 0 and one group at ZT 12 for 4 successive days. A separate group with no exposure to TMT was also included as a control. Animals were sacrificed one day after the last exposure to TMT, and PER2 and c-Fos protein were quantified in the SCN, amygdala, hippocampus, and piriform cortex. Exposure to TMT had a strong effect at ZT 0, decreasing PER2 expression at this time point in most regions except the SCN, and reversing the normal rhythm of PER2 expression in the amygdala and piriform cortex. These changes were accompanied by increased c-Fos expression at ZT0. In contrast, exposure to TMT at ZT 12 abolished the rhythm of PER2 expression in the amygdala. In addition, increased c-Fos expression at ZT 12 was only detected in the central nucleus of the amygdala in the TMT12 group. TMT exposure at either time point did not affect PER2 or c-Fos in the SCN, indicating that under a light-dark cycle, the SCN rhythm is stable in the presence of repeated exposure to a fear-inducing stimulus. Taken together, these results indicate that entrainment to a fear-inducing stimulus leads to changes in PER2 and c-Fos expression that are detected 24 hours following the last exposure to TMT, indicating entrainment of endogenous oscillators in these regions. The observed effects on PER2 expression and c-Fos were stronger during the early day than during the early night, possibly to prepare appropriate systems at ZT 0 to respond to a fear-inducing stimulus.

Mixture of cis-3-hexenol and trans-2-hexenal attenuates behavioral and stress responses induced by 2,5-dihydro-2,4,5-trimethylthiazoline and electric footshock stress in rats

Green odor (GO), a mixture of cis-3-hexenol and trans-2-hexenal, attenuates stress responses and anxiety to psychological stressors in rodents; however, it remains unknown whether GO affects behavioral and stress responses to risk-related olfactory stimuli and actual noxious stimuli. The present study investigated the effects of green odor on behavioral and plasma adrenocorticotropic hormone (ACTH) responses to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a component of fox feces, and electric footshock (FS) stress. When rats were simultaneously exposed to TMT and GO, they showed decreases in immobility and plasma ACTH levels compared with TMT alone. GO exposure after TMT increased immobility, but blocked the elevation of plasma ACTH levels compared with rats exposed to distilled water after TMT. This means that GO presentation during TMT attenuated the TMT-induced behavioral response and GO presentation during and after TMT inhibited TMT-induced elevation of plasma ACTH levels. Furthermore, electric FS-induced plasma ACTH elevations were attenuated by simultaneous GO and FS exposure. GO presentation after FS attenuated plasma ACTH elevations and fecal responses. These findings reveal that GO has alleviating effects on olfactory stimulus- and noxious stimulus-induced behavioral and endocrinal responses.

Inactivation of the prelimbic cortex enhances freezing induced by trimethylthiazoline, a component of fox feces

Previous research has demonstrated that the rodent medial prefrontal cortex (mPFC) is critical for the expression of unconditioned defense behaviors. The prelimbic (PL) and infralimbic (IL) cortices comprise the majority of the mPFC, but the role of these regions in mediating unconditioned defense behaviors is not well understood. In order to address this, we temporarily inactivated the PL or IL and documented the effects of these manipulations on freezing induced by trimethylthiazoline (TMT), a component of fox feces, and center region avoidance in the open field (OF). PL inactivation enhanced TMT-induced freezing, but had no effect on OF behavior. IL inactivation had no effect on any behavioral measure. The results of this study are the first to demonstrate that the PL can have an inhibitory role with regard to unconditioned defense behavior. Further research is needed to define the parameters under which the PL inhibits unconditioned defense behavior.

Infection pressure of human alveolar echinococcosis due to village and small town foxes (Vuples vulpes) living in close proximity to residents

This study investigated the epidemiological and ecological factors to assess the infection pressure of alveolar echinococcosis to human which are living in villages and small towns. Foxes and fox faeces were examined for Echinococcus multilocularis and foxes were observed by radio telemetry in Upper Bavaria, Germany. Forty-three percent of the village foxes (n = 65) had been infected with E. multilocularis. This prevalence rate did not differ significantly from the prevalence among rural foxes, which was 39% (n = 33; χ2 = 0.12, df = 1, p = 0.727) determined by the intestinal scraping technique. PCR analyses of fox faeces showed a higher infection rate of 35% (n = 26) among rural foxes than among foxes in villages and small towns (26%, n = 69; χ2 = 0.68, df = 1, p = 0.411). One quarter of the fox faecal samples come from private gardens of residents. The radio-tracking study on 17 foxes showed that foxes preferred the built-up area and grassland outside the villages. Village foxes concentrated their activity within a range of 500 m around the settlement. Sixty-four percent of all bearings for radio-tracked foxes showed positions in areas outside the town, and 36% of bearings were within the settlement. Village foxes, which are infected with E. multilocularis, are able to carry the parasite continuously into settlements and fox faeces present an immediate source of infection to humans, especially within their gardens. Therefore, foxes are responsible for environmental E. multilocularis egg contamination in the vicinity of humans, leading to an infection risk to inhabitants of villages and small towns.

Assessing carnivore diet by faecal samples and stomach contents: a case study with Alpine red foxes

Abstract Research on the feeding habits of mammalian carnivores relies mainly on the analysis of stomach contents and faecal samples, but the outcomes of these two methods have only been compared in a few studies, with contrasting conclusions. In an Alpine area of NW Italy, we analysed both fox faeces collected along standardised transects, and the stomach contents of road-killed individuals. Faecal analysis involved the identification of macroscopic fragments, the identification of earthworm chaetae, and the assessment of relative volumes using Kruuk and Parish’s technique. Use of both methods indicated that the diet of the red fox included mainly fruit and mammals, but quantitative differences emerged. Garbage, birds, and cultivated fruit were overrepresented in the stomach contents, while earthworms, mammals, and wild fruit prevailed in the faecal samples. Logistic Regression Analysis suggested that the method of analysis was the main factor in determining the occurrence of food items in fox diet. Nonetheless, evidence suggests that road-killed foxes may include a disproportionately high percentage of synanthropic individuals and therefore be biased towards anthropogenic food. Results suggest that by using Kruuk and Parish’s technique, the main limitation of faecal analysis, i.e. the inaccurate estimation of the relative volume of each food item, can be overcome.

Calcitonin gene-related peptide in the bed nucleus of the stria terminalis produces an anxiety-like pattern of behavior and increases neural activation in anxiety-related structures.

Calcitonin gene-related peptide (CGRP) evokes anxiety-like responses when infused into the lateral ventricle of rats. Because the bed nucleus of the stria terminalis (BNST) lies immediately adjacent to the lateral ventricle, is rich in CGRP receptors, and has itself been implicated in anxiety, we evaluated the hypothesis that these effects are attributable to stimulation of CGRP receptors within the BNST itself. Bilateral intra-BNST, but not dorsal, CGRP infusions (0, 200, 400, 800 ng/side) enhanced startle amplitude in a dose-dependent manner, and produced an anxiety-like response on the elevated plus maze. Intra-BNST infusion of the CGRP antagonist, αCGRP(8-37), blocked the effect of CGRP on startle, and also blocked startle potentiation produced by exposure to trimethylthiazoline (a component of fox feces that induces anxiety-like behavior in rats). Intra-BNST, but not dorsal, CGRP infusions also increased c-Fos immunoreactivity in a number of anxiety-related brain areas (central nucleus of the amygdala, locus ceruleus, ventrolateral septal nucleus, paraventricular hypothalamic nucleus, lateral hypothalamus, lateral parabrachial nucleus, dorsal raphe nucleus, and nucleus accumbens shell), all of which receive direct projections from the BNST. Together, the results indicate that the activation of BNST CGRP receptors is both necessary and sufficient for some anxiety responses and that these effects may be mediated by activation of a wider network of BNST efferent structures. If so, inhibition of CGRP receptors may be a clinically useful strategy for anxiety reduction.

Increased BOLD activation to predator stressor in subiculum and midbrain of amphetamine-sensitized maternal rats

Amphetamine, which is known to cause sensitization, potentiates the hormonal and neurobiological signatures of stress and may also increase sensitivity to stress-inducing stimuli in limbic areas. Trimethylthiazoline (5μL TMT) is a chemical constituent of fox feces that evokes innate fear and activates the neuronal and hormonal signatures of stress in rats. We used blood oxygen level dependent (BOLD) MRI to test whether amphetamine sensitization (1mg/kg, i.p. ×3days) in female rats has a lasting effect on the neural response to a stress-evoking stimulus, the scent of a predator, during the postpartum period. The subiculum and dopamine-enriched midbrain VTA/SN of amphetamine-sensitized but not control mothers showed a greater BOLD signal response to predator odor than a control putrid scent. The greater responsiveness of these two brain regions following stimulant sensitization might impact neural processing in response to stressors in the maternal brain.

Responses of naïve and experienced European rabbits to predator odour

The response of prey species to predator scent has been investigated in many mammalian species; however, there is little information about the responses of European wild rabbits at the population level. Therefore, we conducted a simple experiment to investigate the behavioural response of a rabbit population to native predator cues in the wild. We compared the response to the scent of a predator (red fox) in a wild rabbit population bred in semi-natural conditions and naive to terrestrial predators with the response of a population in a similar environment where terrestrial predators were present. The response to predators was based on rabbit abundance, inferred from pellet counts and measured by the defecation rate per day (DRD). Our results indicate that rabbits responded to the odour of fox faeces in the treatment warrens, resulting in a lower DRD. The main anti-predator behaviour observed was spatial avoidance (warren abandonment), which seemed to be more accentuated for rabbits who had not previously had contact with foxes in the plot where terrestrial predators were excluded. In both the fenced and the unfenced plot, the differences in the effect of the predator odour between the control and treatment warrens disappeared after cessation of treatment, suggesting a flexible and adaptive behaviour of rabbits to predator cues.

Fox defecation behaviour in relation to spatial distribution of voles in an urbanised area: An increasing risk of transmission of Echinococcus multilocularis?

Urbanisation of alveolar echinococcosis is a new phenomenon that has been highlighted during the last few decades. It has thus become necessary to understand the dynamics of transmission of Echinococcus multilocularis in urbanised areas. Spatial heterogeneity of infection by E. multilocularis has been explained as the result of a multifactorial dependence of the transmission in which the factors depend on the scale of the investigation. The aim of this study was to assess, in an urbanised area, the effect of such environmental factors as season, habitat type and the level of urbanisation, on the availability of two major intermediate hosts (Microtus spp. and Arvicola terrestris), the distribution of red fox faeces and the distribution of E. multilocularis as determined by detection of coproantigens in faeces. Results of the study revealed higher densities of Microtus spp. in rural than in peri-urban areas. Moreover this species was highly aggregated in urban wasteland. Arvicola terrestris densities did not appear to be linked to the level of urbanisation or to the type of habitat studied. Distribution of faeces was positively linked to distance walked and to Microtus spp. and A. terrestris distributions whatever the level of urbanisation. Such a distribution pattern could enhance the transmission cycle in urban areas. The Copro-ELISA test results on faeces collected in the field revealed that ODs were significantly negatively correlated with the abundance of A. terrestris. The larger population densities of Microtus spp. found in urban wastelands and the well known predominance of Microtus spp. in the red fox diet in the region suggest that Microtus spp. may play a key role in urban transmission of the parasite in the study area.

Identification of the Tibetan fox (Vulpes ferrilata) and the red fox (Vulpes vulpes) by copro‐DNA diagnosis

Studies on feeding habits based on faeces dissecting are imperative to understand the natural history of Tibetan foxes and their functions in the transmission of a lethal zoonotic parasite, Echinococcus multilocularis. However, Tibetan foxes and red foxes live sympatrically on the Tibetan plateau, China. Therefore, the faeces of Tibetan foxes must be distinguished from those of red foxes. We established a diagnostic method to distinguish the faeces of the two species by amplifying a portion of the mitochondrial cytochrome b gene (cytb) and digesting with the restriction enzymes BamHI and SspI, to produce specific diagnostic banding patterns. This PCR-RFLP assay enabled rapid, accurate and easily performed identification and differentiation of the two species.

Determination of bromadiolone residues in fox faeces by LC/ESI-MS in relationship with toxicological data and clinical signs after repeated exposure

In many countries, the fox ( Vulpes vulpes), predator of small mammals, is particularly affected by anticoagulant rodenticides such as bromadiolone due to secondary poisoning. Nevertheless, to date, no method of exposure monitoring is applicable in the field over large areas, and no toxicological data are available concerning sensitivity of foxes to bromadiolone. The aim of this work was to compare excretion kinetics of bromadiolone in fox faeces with clinical and haemostatic effects after repeated exposure to intoxicated voles. A sensitive method for the quantification of bromadiolone excretion in fox faeces and plasma was developed, using liquid chromatography combined with electrospray ionisation mass spectrometry (LC/ESI-MS). The LoD was 0.9 μg/kg and 0.15 μg/L, and the LoQ was 3.0 μg/kg and 0.5 μg/L, in faeces and in plasma, respectively. Four captive foxes were fed for 2 or 5 days with water voles ( Arvicola terrestris Sherman) spiked with bromadiolone at concentrations close to those measured in the field. Faeces and blood were collected for bromadiolone titration, and blood-clotting tests were performed to monitor fox health daily during 10 days and then every 3–4 days until the end of the experiment (D28). Then, after euthanasia, a complete necropsy was performed, and levels of bromadiolone residues in the liver were determined. Bromadiolone residues were detected in faeces 15 h after the first exposure. They increased dramatically during the exposure period and then gradually decreased, but they remained detectable at the end of the experiment, i.e., 26 days after the last exposure. Bromadiolone residues in plasma showed a similar pattern but were no longer detectable 7–24 days after the last exposure. Two foxes presented very severe external haemorrhages, requiring the administration of the antidote vitamin-K1. Bromadiolone residues in faeces and their relationships with exposure and other direct-markers that were measured are discussed. Liver residues and the toxicity data of our study will help to interpret data from fox carcasses collected by wildlife disease surveillance networks. These findings provide a basis for programs aiming to monitor the exposure of wild fox populations to bromadiolone using non-invasive methods based on standard sampling and analysis of residues in faeces.

Cave-dwelling bats do not avoid TMT and 2-PT – components of predator odour that induce fear in other small mammals

Recognition and avoidance of predators is fundamental for the survival of prey animals. Here we conducted the first study assessing chemosensory predator recognition in cave-dwelling bats. We used a Y-maze approach to test the reaction of greater mouse-eared bats (Myotis myotis) to two synthetically derived components of predator odour (2,4,5-trimethyl-3-thiazoline, TMT, a component of fox faeces scent; and 2-propylthietane, 2-PT, a component of mustelid scent) and to the natural scent of the least weasel (Mustela nivalis). It is well documented that rodents and several other small mammals show strong and at least partly innate fear reactions when confronted with these odorants. By contrast, the bats did not show any avoidance or fear reaction, despite the fact that relatively high odorant concentrations were presented. Furthermore, they did not react differently towards predator scent and towards acrid but otherwise neutral odours (basil or goat). The number of entries into in the Y-maze arm with the odour source and the time spent in this arm as well as the bats' overall exploratory activity and several other behavioural variables were not affected by the odour treatments. Generally, the sense of smell is well developed in bats and plays an important role in bat behavioural ecology. It is thus somewhat surprising that the bats did not show any avoidance reaction to predator scent, even though direct contact with a mustelid or fox would result in death. We discuss ecological explanations that might have prevented bats from evolving olfactory predatory recognition and avoidance.

Exposure to TMT odor affects adrenal sympathetic nerve activity and behavioral consequences in rats

The odor of 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a synthetic compound isolated from fox feces, induces various emotional behavioral and stress responses. Here we examined the effect of TMT on behavioral responses and adrenal sympathetic nerve activity (ASNA) in rats. TMT increased freezing behavior, defensive-burying and defensive-attack, and decreased exploration, grooming and approach behaviors. On the other hand, butyric acid (BA), a pungent but non-predatory odor, increased defensive-burying only. TMT increased ASNA strongly, whereas the effects of BA increased ASNA extremely weakly. Furthermore, pre-treatment with the histaminergic H1-receptor antagonist diphenhydramine eliminated the effects of TMT on ASNA. These findings suggest that TMT odor affects autonomic neurotransmission via histaminergic neurons. Exposure to TMT odor likely regulates the controlling autonomic function and output to a motor system simultaneously, evoking behavioral stress responses.

Spatial pattern of adult trees and the mammal‐generated seed rain in the Iberian pear

The degree to which plant individuals are aggregated or dispersed co‐determines how a species uses resources, how it is used as a resource, and how it reproduces. Quantifying such spatial patterns, however, presents several methodological issues that can be overcome by using spatial point pattern analyses (SPPA). We used SPPA to assess the distribution of P. bourgaeana adult trees and their seeds (within fecal samples) dispersed by three mammals (badger, fox, and wild boar) within a 72‐ha plot across a range of spatial scales. Pyrus bourgaeana trees in our study plot (n=75) were clearly aggregated with a critical spatial scale of ca 25 m, and approximately nine randomly distributed tree clusters were identified. As expected from their marking behaviors, the spatial patterns of fecal deposition varied widely among mammal species. Whereas badger feces and dispersed seeds were clearly clustered at small spatial scales (<10 m), boar and fox feces were relatively scattered across the plot. A toroidal shift null model testing for independence indicated that boars tended to deliver seeds to the vicinity of adult trees and thus could contribute to the maintenance and enlargement of existing tree clusters. Badgers delivered feces and seeds in a highly clumped pattern but unlike boars, away from P. bourgaeana neighborhoods; thus, they are more likely to create new tree clusters than boars. The strong tree aggregation is likely to be the result of one or several non‐exclusive processes, such as the spatial patterning of seed delivery by dispersers and seedling establishment beneath mother trees. In turn, the distinctive distribution of P. bourgaeana in Doñana appeared to interact with the foraging behavior of its mammalian seed dispersers, leading to neighbourhood‐specific dispersal patterns and fruit‐removal rates. Our study exemplifies how a detailed description of patterns generates testable hypotheses concerning the ecology of zoochorous. Pyrus bourgaeana dispersers were unique and complementary in their spatial patterning of seed delivery, which likely confers resilience to their overall service and suggests lack of redundancy and expendability of any one species.

When the Ghost of Predation has Passed: Do Rodents from Islands with and without Fox Predators Exhibit Aversion to Fox Cues?

AbstractAnti‐predator behavior can alter the dynamics of prey populations, but little is known about the rate at which anti‐predator behavior is lost from prey populations following predator removal. The Channel Islands differ in whether they have historically contained a top predator, the Island Fox (Urocyon littoralis), in evolutionary time (approximately 6200–10 000 yr). On a historically fox‐containing island and two historically fox‐free islands in 2007, I deployed live traps that contained olfactory cues of fox predators (fox feces), olfactory cues of an herbivore (horse feces) or a no‐feces control. Due to a captive breeding program, foxes on the historically fox‐containing island were effectively removed from 1998 to 2004. Rodents from one of the historically fox‐free islands did not respond to fox cues, whereas rodents on the historically fox‐containing island were more likely to be captured in a control trap and less likely to be captured in a fox‐cue trap. Results from the other historically fox‐free island that experienced a recent population bottleneck and period of captive rearing exhibited a preference for horse‐scented traps. These results suggest that, on islands where foxes are the primary predators, anti‐predator behavior in response to olfactory cues is not likely to be rapidly lost by short‐term removals of foxes, although the nature of anti‐predator behavior may depend upon founder events and recent population dynamics (e.g. population bottlenecks or several generations in captivity).

Comparative Fear-Related Behaviors to Predator Odors (TMT and Natural Fox Feces) before and after Intranasal ZnSO4 Treatment in Mice

The possibility that synthetic 2,4,5-trimethylthiazoline (TMT), frequently used to induce unconditioned fear in rodents, could be more a pungent odor activating intranasal trigeminal nerve fibers rather than a predator odor index is currently discussed. In order to explore this question, the present study compared fear-related behaviors to predator odors (synthetic 10% TMT and natural fox feces) and toluene (as an irritant compound without ecological significance) before and after intranasal ZnSO4 perfusion which is known to provoke transient anosmia. Results show that natural fox feces could be consider as a pure olfactory (CN I) nerve stimulant while 10% TMT appeared to be a mixed olfactory (CN I) and trigeminal (CN V) nerves stimulant with a great olfactory power and a low trigeminal power. These findings suggest that behavioral neuroscience studies should use concentrations lower than 10% TMT to obtain fear-related behaviors similar to those obtained with natural fox feces odor.

Small-mammal consumption by sympatric canids in southern Brazil: niche overlap and prey selection

The crab-eating fox, Cerdocyon thous, and the pampas fox, Lycalopex gymnocercus, are two endemic canids from the neotropics that are sympatric in the Rio Grande do Sul State, southern Brazil. Previous studies indicated that both species feed mainly on small mammals, without marked differences between their diets, at least when small-mammal genera are not determined. In the present study, our objective was to evaluate the similarity in diet composition of those foxes, identifying the genera of small mammal consumed. Besides that, we also investigated potential patterns of prey selection by the canids. We analyzed 222 samples of fox feces from the National Park of Aparados da Serra (NPAS) in southern Brazil (29°10’S, 50°05’W) collected from 2000 to 2005. Fox species were determined by identification of hairs found in scats. We also conducted, between 2000 and 2002, a survey of small mammals using capture-mark-recapture techniques. Our results indicated that the species studied do not differ significantly in relation to the small mammals they consume. We also detected that both canids seem to prefer feeding on rodents of the genus Akodon. On the other hand, rodents of the genus Oligoryzomys were consumed less than would be expected. Our results indicated that prey similarity is very high for the studied canids, even under a more refined analysis of small-mammal preys. This pattern supports the suggestion that niche differentiation between C. thous and L. gymnocercus probably occurs through other mechanisms than diet (e.g. habitat or activity time). Key words: araucaria forest, cerdocyon thous, rodents, feeding habits, niche overlap, lycalopex gymnocercus.

Seed dispersal of Solanum thomasiifolium Sendtner (Solanaceae) in the Linhares Forest, Espírito Santo state, Brazil

The aim of this study was to analyse seed dispersal and establishment of Solanum thomasiifolium in an area of "nativo" vegetation in Espirito Santo state on the southeastern Brazilian coast. Ten species of birds, the crab-eating fox (Cerdocyon thous), and one species of lizard (Tropidurus torquatus) fed on S. thomasiifolium fruits and dispersed viable seeds in their faeces. The proportional contribution of each of these groups to seed dispersal was 77% (birds), 19% (crab-eating fox) and 4% (lizards). Ants also contributed to seed dispersal. More seeds were deposited in vegetation islands than in the surrounding open areas. Germination rates of seeds collected directly from fruit (control), bird droppings, the faeces of crab-eating foxes and lizards were, respectively, 64, 64, 53, and 80 %. Differences among these rates were all significant, except between birds and control. Lizards were important as seed carriers between nearby islands and they expelled a higher proportion of viable seeds. Birds and the crab-eating foxes did not enhance seed germination, but promoted seed dispersal over a wider area. Plant architecture, fruit productivity, fruit characteristics and the diversity of frugivores are important for the success of S. thomasiifolium in habitat colonization.

Multiplex PCR system for identifying the carnivore origins of faeces for an epidemiological study on Echinococcus multilocularis in Hokkaido, Japan

A multiplex PCR system was developed to identify the carnivore origins of faeces collected in Hokkaido, Japan, for epidemiological studies on Echinococcus multilocularis. Primers were designed against the D-loop region of mitochondrial DNA. Two separate primer mixtures (mix 1, specific forward primers to fox, raccoon dog and dog, and a universal reverse primer [prH]; and mix 2, specific forward primers to cat, raccoon and weasels and prH) were used so that the PCR products (160 bp, fox and cat; 240 bp, raccoon dog and raccoon; and 330 bp, dog and weasel) were distinguished by size. The multiplex PCR exhibited no cross-reactivity between carnivore species and did not amplify DNA from rodent prey. When 270 field-collected faeces were examined, 250 showed single PCR products belonging to specific target sizes, suggesting successful carnivore identification for 92.6% of samples. Taeniid eggs were detected in 11.1% of samples and coproantigen in 30.4%; whereas the prevalences of taeniid eggs and coproantigen were 12.9% and 34.0% in fox faeces, and 0% and 26.3% in cat faeces, respectively. These results suggest that the prevalence in different target animals can be evaluated individually and precisely using multiplex PCR system.