Application of a formulation of the nematophagous fungus <Emphasis Type="Italic">Duddingtonia</Emphasis><Emphasis Type="Italic">flagrans</Emphasis> in the control of cattle gastrointestinal nematodiosis

The viability of a formulation of Duddingtonia flagrans was assessed in the control of parasite gastrointestinal nematodes of cattle. Two groups (A and B) of eight crossbred Holstein × Zebu cattle, approximately one year old, were placed in Brachiaria decumbens pasture. Each animal in group B (treated) received orally 20 g sodium alginate pellets containing mycelial mass of the D. flagrans fungus, while the animals in the group A (control) received pellets without fungus for seven months, starting in March 2005. The egg per gram of feces counting the gastrointestinal nematodes showed a difference (P < 0.05) in the treated group in June, July and August, with reductions of 58% (June), 47% (July) and 51% (August) compared to the control group. The infective larvae recovered in the pastures collected up to 20 cm from distance of the fecal dung in group B differed (P < 0.01) from the larvae recovered in group A. At the end of the experimental period, the animals in group B presented a greater weight gain (P < 0.01) compared to the untreated group (A). The treatment of cattle with pellets containing the D. flagrans nematophagous fungus, at the dose and duration used was effective in controlling the infective larvae of gastrointestinal nematodes of cattle.     

Effects of times and storage conditions of Duddingtonia flagrans chlamydospores in sodium alginate pellets on its nematode predatory ability

This study aimed to determine the effects of Duddingtonia flagrans contained in sodium alginate pellets on trichostrongylide larvae under different storage conditions and durations. The in vitro predatory activity of D. flagrans in pellets against trichostrongylide larvae in sheep faeces were assessed at 0, 1, 3, 6, 9, and 12 months after the pellet was stored under four different conditions (i.e. ?20, 4°C, outdoors, and indoors). These results revealed that the numbers of larvae in faeces of sheep treated with pellets containing chlamydospores (treatment groups) were significantly lower than those in the control groups (without chlamydospores) for all trial months under four storage conditions for different durations (p?<?.05). The obtained reduction rates of the infective larvae (L3) in the four treatment groups ranged from 45.62% to 96.73% throughout the entire experiment. The overall mean L3 reduction percentages were 89.22%?±?3.74%, 88.97%?±?1.33%, 68.60%?±?14.31%, and 75.45%?±?13.18% for 4°C refrigeration, ?20°C refrigeration, indoor, and outdoor conditions, respectively. The pellets stored under these storage conditions for a year were provided to sheep for ingestion (in vivo test), and the results showed that the number of recovered larvae in sheep faeces at 24?h after ingestion were significantly lower than that before ingestion. For in vivo test, the L3 reduction percentage in the faeces was 90.99% (?20°C), 74.81% (outdoor), 83.53% (4°C), and 65.60% (indoor). Under the four storage conditions, D. flagrans spores contained in the pellets can maintain their survival ability to a varying degree in a year.     

Biological control on gastrointestinal nematodes in cattle with association of nematophagous fungi

This study aimed to evaluate the efficacy of the association of the nematophagous fungi (Duddingtonia flagrans-AC001); (Pochonia chlamydosporia-VC4) and (Arthrobotrys robusta-I31) in a pelletised formulation of a sodium alginate matrix. The viability and activity of pellet germination and fungal activity (after encapsulation) were evaluated using in vitro and in vivo tests. Next, 12 heads of cattle, Dutch mestizo x zebu, with an average age of 12 months were dewormed with an anthelmintic. Next, 20 days after treatment with the anthelmintic, the animals were randomly divided into two groups of six animals each, and placed in two paddocks with 7.0?ha each of Brachiaria decumbens with historical grazing by animals naturally infected by gastrointestinal nematode parasites. At first, each animal was treated with 2?g of pellets per 10?kg of animal, containing the associated fungi (AC001?+?VC4?+?I31) administered twice a week in conjunction with commercial feed. Each animal in the control group received 2?g of pellets without mycelia added to the feed. The percentage reduction of infective larvae in the in vitro test was 94% (p?in vivo test, the treated animals with fungal association had lower egg counts per gram of faeces (p?相似文献   

Influence of the preservation period in silica-gel on the predatory activity of the isolates of Duddingtonia flagrans on infective larvae of cyathostomins (Nematoda: Cyathostominae)

The continued maintenance of nematophagous fungi predatory activity under laboratory conditions is one of the basic requirements for a successful biological control. The purpose of this study was to evaluate the influence of time on the preservation of the fungus Duddingtonia flagrans (AC001 and CG722) stored in silica-gel for 7 years and their subsequent predatory activity on cyathostomin L₃ larvae in 2% water-agar medium (2% WA). Samples of the isolates AC001 and CG722, originating from vials containing grains of silica-gel sterilized and stored for 7 years, were used. After obtaining fungal conidia, the predation test was conducted over 7 days on the surface of 9.0 cm Petri dishes filled with 2% WA. In the treated groups each Petri dish contained 500 cyathostomin L₃ and conidia of fungal isolates in 2% WA. In the control group (without fungi) the plates contained 500 L₃ in 2% WA. The experimental results showed that isolated AC001 and CG722 were efficient in preying on cyathostomin L₃ (p < 0.01) compared to control (without fungus). However, no difference was observed (p > 0.01) in the predatory activity of the fungal isolates tested. Comparing the groups, there was a significant reductions of cyathostomin L₃ (p < 0.01) of 88.6% and 78.4% on average recovered from the groups treated with the isolates AC001 and CG722, respectively, after 7 days. The results of this test showed that the fungus D. flagrans (AC001 and CG722) stored in silica-gel for at least 7 years maintained its predatory activity on cyathostomin L₃.     

An extracellular serine protease of an isolate of Duddingtonia flagrans nematophagous fungus

This study aimed to present a protease produced by Duddingtonia flagrans fungus (AC001), and to evaluate its activity in the biological control of cyathostomin infective larvae (L₃). The crude extract from D. flagrans grown in liquid medium was applied first to a DEAE-Sepharose? and later to a CM-Sepharose? ion exchange column. Protease activity was determined under different pHs and temperatures. Subsequently, the effects of metal ions and phenylmethylsulfonyl fluoride (PMSF) inhibitor on activity were evaluated. Next, the protease activity in the biological control of nematodes was tested. A new 38 kDa serine protease (Df1) was purified. Optimum activity was obtained at pH 8.0 and 60°C; CuSO₄, ZnSO₄ and PMSF strongly inhibited the activity. Df1 (AC001) showed an L₃ reduction rate of 58%. In conclusion, a serine protease produced by D. flagrans (AC001) has been isolated, which is effective in the in vitro destruction of cyathostomin L₃.     

In vitro association of nematophagous fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34) and Pochonia chlamydosporia (VC1) to control horse cyathostomin (Nematoda: Strongylidae)

In vitro effects of nematophagous fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34) and Pochonia chlamydosporia (VC1) were evaluated against eggs and third-stage infective larvae (L₃) of horse cyathostomin (Nematoda: Strongylidae). The following percentage reductions compared with the control group were observed after a 20-day exposure period: AC001, 61.6%; NF34, 66.1%; VC1, 73.2%; group AC001 + VC1, 86.8%; NF34 + VC1, 77.3%; AC001 + NF34, 92.4%. The results showed that the fungal isolates (VC1, AC001 and NF34), acting alone or in conjunction, were efficient in controlling horse cyathostomin under in vitro conditions.     

Use of statistical tools in the study of the conditions of predation of Duddingtonia flagrans versus Panagrellus sp

The objective of the present work was to study the conditions of predation of Duddingtonia flagrans conidia versus Panagrellus sp using response surface methodology. Conidia of D. flagrans (AC001) isolate were transferred into water-agar (WA) culture media at different pHs and different concentrations defined according to Central Composite Design (CCD). Five different concentrations of D. flagrans conidia: (1292, 500, 1000, 1500 and 1707) were used. For 2%WA media were used the following pHs were used: 6.29, 6.5, 7.0, 7.5 and 7.71. The response of the design corresponded to the number of larvae (transformed to square root scale) observed at the end of the experiment (10 days). At the tenth day, the non predated larvae were recovered in the Petri dishes. The results showed that 2%WA media at pH 7.0 contributed to improve the predatory activity of conidia of D. flagrans, and therefore this tool may be used in future studies under laboratory and natural conditions.     

Larvaler «Triähanismus» Und die Evolution alternativer Entwicklungszyklen bei einem saprobionten Nematoden: Rhabditis (Pelodera) orbitalis (Nematoda) als Larvalparasit im Auge von Mäusen1

Larval triphenism and the evolution of alternative life-cycles in a microbotrophic nematode: Rhabditis (P.) orbitalis – a larval parasite in the eye orbits of murid rodents Along with the normal third stage larva and the resistant dauerlarva, Rh. orbitalis posesses an infective larva, which is an obligate parasite in the eye orbits of mice and voles. Dauerlarvae as well as infective larvae develop from a common pre-stage («girdle-larva»), determined by the quality of food stored in the intestinal cells. – Dauerlarvae may tolerate desiccation (at room-temperature and -humidity) up to 4 weeks. If stored in tap water (at 6°C), they will survive for some month being able to restore development. – Infective larvae never survive desiccation but can also be stored in tap water at low temperatures. Food uptake from the lachrymal fluid of a host is needed prior to restoring development. The phenomenon involved is referred to as a necessary adaptation to the ecological conditions in the rodents' nests. It is also pointed out to the different strategies of other parasitic nematode species.     

Establishment of Arthrobotrys flagrans as biocontrol agent against the root pathogenic nematode Xiphinema index

Plant-parasitic nematodes cause devastating agricultural damage worldwide. Only a few synthetic nematicides can be used and their application is limited in fields. Therefore, there is a need for sustainable and environment-friendly alternatives. Nematode-trapping fungi (NTF) are natural predators of nematodes. They capture and digest them with their hyphae and are starting to being used as bio-control agents. In this study, we applied the NTF Arthrobotrys flagrans (Duddingtonia flagrans) against the wine pathogenic nematode Xiphinema index. A. flagrans reduced the number of X. index juveniles in pot cultures of Ficus carica, an alternative host plant for X. index, significantly. Sodium-alginate pellets with A. flagrans spores were produced for vineyard soil inoculation under laboratory conditions. The NTF A. conoides, A. musiformis and A. superba were enriched from several soil samples, showing their natural presence. Trap formation is an energy-consuming process and depends upon various biotic and abiotic stimuli. Here, we show that bacteria of the genus Delftia, Bacillus, Pseudomonas, Enterobacter and Serratia induced trap formation in NTF like A. conoides and A. oligospora but not in A. flagrans in the absence of nematodes. The application of NTF along with such bacteria could be a combinatorial way of efficient biocontrol in nematode-infested soil.     

Mechanical production of pellets for the application of entomopathogenic nematodes: effect of pre-acclimation of Steinernema glaseri on its survival time and infectivity against Phyllophaga vetula

The low survival time and diminished infectivity by entomopathogenic nematodes (EPNs) from granular formulations limit their efficiency against agricultural insect pests. This study determined the benefit of pre-acclimating infective juveniles (IJs) of Steinernema glaseri (NJ-43 strain) on extending their mean survival time (ST_m) in diatomaceous earth (DE) pellets and increasing their infectivity against Phyllophaga vetula. The IJs were reared in Galleria mellonella larvae placed in Petri dishes containing plaster of Paris (PP) or modified White traps (WTs). Pelletisation was performed in a machine operating on the principle of laminar flow using DE Celite® 209. Pellets were stored at room temperature (23?±?3°C) and high relative humidity (96–100%). IJs harvested from WTs between the 3rd and the 5th days after the onset of emergence were more infective on P. vetula and pre-acclimation of S. glaseri in PP increased significantly its ST_m in the pellets; from 23.1 to 34.5 days, compared with non-pre-acclimatised IJs from WTs. However, juveniles with or without pre-acclimation formulated in DE pellets failed to achieve significant control of P. vetula. These results are discussed in light of the relationship between EPN survival and host infection by EPNs with possible effects of the formulation in DE pellets.     

Factors responsible for transition of the <Emphasis Type="Italic">Duddingtonia flagrans</Emphasis> carnivorous fungus from the saprotrophic to the zootrophic nutrition type

Quantitative investigation of the factors responsible for trap formation in the nematophagous fungus Duddingtonia flagrans F-882 in submerged liquid culture was carried out. The data obtained suggest a complex program for the regulation of zootrophic nutrition in D. flagrans. Optimal concentrations of such carbon and nitrogen sources as sucrose (0.4%), ammonium ions (0.2%), and tryptone (0.2%) promote trap formation in the case of contact with the nematodes Panagrellus redivivus. Increased concentrations of these compounds, however, inhibit trap formation. The sensitivity of the mycelium to nematode excreta depends on the state of the culture and is increased under limitation by certain nutrient components or in the course of prolonged starvation. A direct correlation was found between the number of caught nematodes and the number of chlamydospores formed on the mycelium. The nutrients obtained from the nematode biomass are used for formation of additional chlamydospores (on average, about 20 chlamydospores per nematode). Environmental and evolutionary aspects of the role of zootrophic nutrition in carnivorous fungi are discussed.     

Influence of different storage methods on the predatory capacity of the fungi Arthrobotrys robusta and Monacrosporium thaumasium after passage through the bovine gastrointestinal tract

The biological control of helminth parasites of bovines by nematophagous fungi is an alternative to the use of drugs with the principal objective of reducing the source of infection available on pastureland. The maintenance of predatory activity of the fungal isolates is one of the basic prerequisites to ensure the success of this form of control. In this study behaviour of the isolates I31 of Arthrobotrys robusta and NF34a of Monacrosporium thaumasium was investigated following three storage methods: stored at 4 °C, cryopreserved with or without cryoprotectants or preserved in silica gel. All samples were subsequently passed through the gastrointestinal tract of calves. The latter involved the administration of 20 g of mycelia to the animals. This quantity was sufficient to recover fungal material from the faeces. The peak reduction in the number of infective larvae in the faeces occurred 24 h after administration of the samples (P < 0.05). The storage at 4 °C was the treatment that produced the greatest reduction in larvae for NF34a (81.3%) and I31 (65.1%) isolates. Nf34a isolate was responsible for the highest percentage reduction of larval helminth populations (P < 0.05). Cryopreservation appears to be an efficient method of preserving isolates, although diminished predatory capacity compared to storage at 4 °C was seen only for isolate NF34a (73.2%). Cryopreservation did not interfere in predatory activity of I31 isolate (P < 0.05). Maintenance of isolates in silica gel showed the lowest reduction throughout the experiment (P < 0.05).     

In vitro Storage of Strawberry and Raspberry in Calcium-Alginate Beads at 4°C

Three-millimeter-long shoot tips of strawberry 'Senga Sengana' and raspberry 'Norna' encapsulated in calcium alginate were stored in vitro at 4 °C in the dark. The cultures which were donors for the shoot tips were grown before encapsulation on shoot multiplication media (Boxus medium with 2.2 µM BAP and 2.46 µM IBA for strawberry, and MS medium with NH₄NO₃ and KNO₃ reduced by 50%, and with 3.55 µM BAP and 0.49 µM IBA for raspberry) as well as on these media supplemented with 10 g l^–1 mannitol or paclobutrazol (1.7 µM for strawberry and 3.4 µM for raspberry). Sodium alginate was dissolved in water, water with sugar or in a culture medium without growth regulators. Regrowth ability of the stored explants and in vitro multiplication in three successive subcultures were evaluated. The encapsulated shoot tips could be stored for 9 months in beads containing sugar or a culture medium. The pre-conditioning of the donor cultures on a mannitol containing medium was beneficial for regrowth ability. The multiplication rate of strawberry and raspberry shoots in the first subculture after storage was lower than that of non-stored cultures. Particularly low multiplication was obtained for strawberry which had been stored for 9 months and for raspberry stored for 3 and 6 months, in combinations where the beads were prepared by dissolving sodium alginate in water. Multiplication of strawberry in the second subculture was generally higher than in non-stored cultures, but multiplication of raspberry was lower also in the second subculture, with the exception of the combination stored for 9 months and pre-cultured on mannitol. In the third subculture, shoot multiplication in both species was similar to that in non-stored cultures.     

Effect of moisture evaporation from diatomaceous earth pellets on storage stability of Steinernema glaseri

Induction of anhydrobiosis and storage stability of entomopathogenic nematodes are influenced by moisture availability. Decreasing moisture content in diatomaceous earth (DE) pellets containing the Steinernema glaseri NJ-43 strain and its effect on survival time and infectivity of the nematode were determined. Pelletisation was performed in a vortex mixer, using DE Celite® 209 as the desiccant material. Pellets were stored at room temperature (23?±?2°C) and high relative humidity (96–100%). Nematode survival and infectivity against last instar greater wax moth, Galleria mellonella, were tested daily. Initial average and average equilibrium moisture content in pellets were 66.7% and 13.6%, respectively, and the infective juveniles mean survival time was 8.8 days. A moisture transfer model based on diffusion and evaporation was evaluated to predict moisture fluctuations within the pellets. We concluded that 84% of variation in S. glaseri infectivity on G. mellonella larvae was explained by the survival of the nematode, whereas 52% of variation in S. glaseri survival was explained by the loss of moisture from the pellets. The moisture transfer model achieved 78% reliability in predicting moisture content and fluctuations. Therefore, the mechanisms of moisture diffusion and evaporation from the surface to the surrounding atmosphere contribute significantly to moisture loss from the pellets.     

Production and partial characterization of Duddingtonia flagrans (AC001) crude extract and its in vitro larvicidal action against trichostrongylid infective larvae

The production and partial characterization of Duddingtonia flagrans (AC001) crude extract and its in vitro larvicidal action against trichostrongylid infective larvae from sheep were studied. D. flagrans was grown in liquid medium with glucose, casein, bibasic potassium phosphate (K₂HPO₄), magnesium sulfate (MgSO₄), zinc sulfate (ZnSO₄), ferrous sulfate (FeSO₄), and copper sulfate (CuSO₄). The proteolytic activity was measured within varied pHs and temperatures. To determine the thermostability, the crude extract was incubated at 28°C for 72 h. To study the effect of different chemical compounds on the activity of the crude extract, the samples were incubated in solutions containing (10 mM): calcium chloride (CaCl₂), copper II sulfate (CuSO₄), zinc sulfate (ZnSO₄), magnesium sulfate (MgSO₄), inhibitor phenylmethylsulfonyl fluoride (PMSF), and 0.5% SDS. Results showed that the highest activity obtained (79.23 U/mL) was at pH 9.0, while the optimum temperature was 60°C (119.6 U/mL). The thermostability analysis demonstrated that after 72 h the activity was maintained or increased. It was found that the CuSO₄, ZnSO₄, and PMSF strongly inhibited the proteolytic activity. Moreover, the MgSO4 and SDS, caused a weak inhibition of the proteolytic activity. There was a significant (P<0.01) reduction in number of treated L₃ when compared to control (94.2%). The results suggest that the crude extract produced by D. flagrans (AC001) in liquid medium exerted larvicidal activity on trichostrongilid L₃ and therefore may contribute to a large-scale industrial production.     

Isolation of nematophagous fungi from eggs and females of Meloidogyne spp. and evaluation of their biological control potential

Fungi were isolated from Meloidogyne spp. eggs and females on 102 field-collected root samples in China. Of the 235 fungi isolated (representing 18 genera and 26 species), the predominant fungi were Fusarium spp. (42.1% of the isolates collected), Fusarium oxysporum (13.2%), Paecilomyces lilacinus (12.8%), and Pochonia chlamydosporia (8.5%). The isolates were screened for their ability to parasitise Meloidogyne incognita eggs in 24-well tissue culture plates in two different tests. The percentage of eggs parasitised by the fungi, the numbers of unhatched eggs and alive and dead juveniles were counted at 4 and 7 days after inoculation. The most promising fungi included five Paecilomyces isolates, 10 Fusarium isolates, 10 Pochonia isolates and one Acremonium isolate in test 1 or test 2. Paecilomyces lilacinus YES-2 and P. chlamydosporia HDZ-9 selected from the in vitro tests were formulated in alginate pellets and evaluated for M. incognita control on tomato in a greenhouse by adding them into a soil with sand mixture at rates of 0.2, 0.4, 0.8 and 1.6% (w/w). P. lilacinus pellets at the highest rate (1.6%) reduced root galling by 66.7%. P. chlamydosporia pellets at the highest rate reduced the final nematode density by 90%. The results indicate that P. lilacinus and P. chlamydosporia as pellet formulation can effectively control root-knot nematodes.     

Feeding rates of common Antarctic gammarid amphipods on ecologically important sympatric macroalgae

Single species feeding trials employing both fresh algal tissues and alginate food pellets containing dried finely ground algal tissues were conducted to examine the relative palatability of sympatric Antarctic macroalgae (three brown and five red macroalgal species) to three common herbivorous gammarid amphipods (Prostebbingia gracilis Chevreux, Gondogeneia antarctica (Chevreux) Thurston, and Metaleptamphopus pectinatus Chevreux). In fresh algal tissue bioassays, both the amphipods P. gracilis and G. antarctica consumed significantly greater amounts of the red alga Palmaria decipiens over all other seven species of macroalgae. The amphipod M. pectinatus failed to consume measurable quantities of fresh thalli of any macroalgae and therefore is likely to feed on other resources. In food pellet bioassays, the consumption rates of amphipods fed with eight different species of macroalgae were compared with consumption rates on a highly palatable control green alga. Alginate pellets containing finely ground tissues of P. decipiens were consistently the most palatable of any of the macroalgae to P. gracilis and G. antarctica, while pellets containing the brown algae Desmarestia menziesii, D. anceps and the red alga Plocamium cartilagineum were not consumed by any of the three amphipod species. Regression analysis indicated that feeding rates of the amphipods P. gracilis and G. antarctica on alginate food pellets were not significantly correlated with known species-specific parameters of macroalgal nutritional quality (%N, %C, C:N ratio, soluble protein, soluble carbohydrate, and lipid). Therefore, differences in amphipod macroalgal palatability are most likely related to other factors including physical and/or chemical deterrents.     

Epizootiology of a nuclear polyhedrosis virus in European spruce sawfly (Gilpinia hercyniae): the rate of passage of infective virus through the gut of birds during cage tests.

The passage of a nuclear polyhedrosis virus (NPV) of the sawfly, Gilpinia hercyniae, through avian gut was studied during cage tests on Sturnus vulgaris (three individuals), Parus ater (one), Parus caerulus (five), and Parus major (one). Following brief infection feeds, polyhedral inclusion bodies of the virus could be detected in bird feces within 0.5 hr. Peak passage of polyhedra occurred in less than 1 hr and none were detected after 2.5 hr. The feces of all birds remained infective (in bioassay tests using first instar G. hercyniae larvae) to the end of the day of infection while those of nine birds remained infective to the next day and of six birds to the third day. One bird, P. major, was also infective on Days 4, 6, and 7. The infectivity of NPV in feces stored for 2 years at +3°C declined by half. Though the scale of their epizootiological contribution is unknown, the comparatively long retention and passage of infective virus suggests birds may be effective in short- and long-distance transport of baculoviruses.     

Survival of Xiphinema index in clay loam soil

A sample of clay loam vineyard soil, containing the nematode Xiphinema index, was divided into equal portions and stored in plastic bags. Nematodes were extracted immediately or remained in fridge for a short time or at room temperature for longer periods. The number of extracted nematodes did not differ significantly between treatments, indicating that X. index in soil samples collected for diagnostic purposes could remain viable for a period up to six months. Four other samples, of similar soil type, were collected from different vineyards and kept stored in plastic bags at room temperature. Variable periods of nematode survival recorded, ranging from less than two years in one sample, up to five years in another one. It is concluded that a long fallow period of at least five years may be required between successive grapevine crops to eliminate the nematode from clay loam soils.     

Disintegration of Dung Pats from Cattle Treated with the Ivermectin Anthelmintic Bolus,or the Biocontrol Agent Duddingtonia flagrans

An experiment was performed during the grazing seasons of 1998, 1999 and 2000 to study the influence of the antiparasitic drug ivermectin and the nematophagous fungus Duddingtonia flagrans on cattle dung disintegration. The faeces originated from groups of animals that were part of a separate grazing experiment where different control strategies for nematode parasite infections were investigated. Each group consisted of 10 first-season grazing cattle that were either untreated, treated with the ivermectin sustained-release bolus, or fed chlamydospores of D. flagrans. Faeces were collected monthly on 4 occasions and out of pooled faeces from each group, 4 artificial 1 kg dung pats were prepared and deposited on nylon mesh on an enclosed pasture and protected from birds. The position of the new set of pats was repeated throughout the 3 years of the study. Each year, the dung pats were weighed 4, 6, 8 and 10 weeks after deposition and immediately afterwards replaced to their initial positions.

Results showed that there was no difference in faecal pat disintegration between groups. However, the time-lag between deposition and complete disintegration of the faeces varied significantly between deposition occasions. Dung pats disappeared within 2 weeks (visual observation) when subjected to heavy rainfall early after deposition, whereas an extended dry period coincided with faeces still remaining 12 months after deposition.