Lorendane Millena de Carvalho

Interaction of the nematophagous fungus Duddingtonia flagrans on Amblyomma cajannense engorged females and enzymatic characterisation of its chitinase

Abstract The present work aimed to evaluate the production and the characterisation of a chitinase from nematophagous fungus Duddingtonia flagrans (AC001) and observe the interaction of this fungus on engorged females of Amblyomma cajennense under laboratory conditions. In assay A, the engorged females of A. cajennense were separated and immersed for 5 seconds in a fungal suspension of 106 conidia/ml of the fungus D. flagrans and placed in Petri dishes, in the dark. In assay B, wheat bran supplemented with 1% chitin and liquid minimal medium was used [K2HPO4 (5.0 g/l), MgSO4 (0.10 g/l), ZnSO4 (0.0050 g/l), FeSO4 (0.001 g/l) e CuSO4 (0.50 mg/l)], as a substrate for chitinase production. To demonstrate the presence of chitinase in the crude extract obtained after the enzymatic extraction, a purification process was developed using a specific adsorption technique. The results from assay A demonstrated the interaction of the D. flagrans conidia produced from chitin-agar on engorged females of A. cajennense. In the assay B, D. flagrans produced a chitinase successfully, with a high value for enzyme activity. The molecular mass of semi-purified enzyme was estimated at approximately 34 kDa. It was concluded that the fungus produced a chitinase and has some entomopathogenic activity, as demonstrated here for the first time; however, it is strongly suggested that further studies are needed to elucidate the molecular mechanism of infection of target organisms by this fungus.

Interaction of the nematophagous fungus Pochonia chlamydosporia and Parascaris equorum eggs in different culture media

Research involving the use of nematophagous fungi in the biological control of parasites of interest to veterinarians has occurred over recent years, with promising results. This article reports the infection of Parascaris equorum eggs by the fungus Pochonia chlamydosporia (isolates VC1 and VC4). Six groups were formed for each isolate, with six different culture media: 2% water–agar (2% WA); agar–chitin (AC); YPSSA (yeast extract, K2HPO4, MgSO4·7H2O, soluble starch); AELA extract (starch + water + agar); 2% corn‐meal–agar (2% CMA); and 2% potato dextrose–agar (2% PDA). A total of 1000 eggs of P. equorum were transferred to each plate containing isolates grown for a period of 7 days (treatment group). Also, 1000 eggs were added to each plate without fungus (controlgroup). The plates were kept in an environmental chamber at 25 °C in the dark for 21 days. After, we analyzed the effects on ovicidal activity: effect 1 (accession shell); effect 2 (penetration hyphae); and effect 3 (destruction of the eggs). No differences were observed in the destruction of eggs between the two isolates. The decreasing effectiveness of the different culture media was: PDA (38.9%); CMA (38.3%); WA (36.7%); YPSSA (36.45%); and AC (32.5%). The highest percentage egg destruction was observed when the strains were grown in culture medium AELA (44.9%); this was the best medium.

First report of the activity of predatory fungi on Angiostrongylus cantonensis (Nematoda: Angiostrongylidae) first-stage larvae☆

The nematode Angiostrongylus cantonensis causes eosinophilic meningoencephalitis in humans and thus alternative methods of control should be studied. The objective of this work was to evaluate the predatory capacity of eight fungal isolates of the species Duddingtonia flagrans (AC001, CG768 and CG722), Monacrosporium thaumasium (NF34), M. sinense (SF53) and Arthrobotrys robusta (I31), A. cladodes (CG719) and A. conoides (I40) on first-stage larvae (L₁) of A. cantonensis under laboratory conditions. The treated groups contained 1000 conidia of the fungal isolates and 1000 A. cantonensis L₁ in Petri dishes containing 2% water-agar medium (2% WA). The control group (without fungi) contained only 1000 A. cantonensis L₁ in 2% WA. Evidence of predation was observed at the end of 7 days. Percentage reductions in L₁ were: AC001, 82.8%; CG768, 71.0%; CG722, 72.8%; NF34, 86.7%; SF53, 89.7%; I40, 48.3%; CG719, 84.7%; and I31, 80.4%. No significant difference was observed (p>0.01) between the actions of the isolates used; however, a difference was noted (p<0.01) in relation to the control group. The results of the present work, confirm previous reports of the effectiveness of the fungi D. flagrans, M. thaumasium, M. sinense and A. robusta in controlling larvae of potentially zoonotic nematodes, this being the first report on A. cantonensis L₁.

Effect of the fungus Pochonia chlamydosporia on Echinostoma paraensei (Trematoda: Echinostomatidae)

Echinostoma paraensei is a trematode of the genus Echinostoma that causes echinostomiasis in humans. The objectives of this study were to: evaluate the ovicidal activity of the nematophagous fungus Pochonia chlamydosporia (VC1 and VC4) on a solid medium 2% water-agar (2% WA) against E. paraensei eggs (assay A); evaluate ovicidal effect (destruction of eggs) of the isolate VC4 in supplemented culture media (assay B); and evaluate the ovicidal ability of the crude extract (VC4) on E. paraensei eggs (assay C). Eggs of E. paraensei (assay A) were placed in Petri dishes containing 2% WA with an isolate of the fungus P. chlamydosporia (VC1 and VC4) grown for 10 days, and without fungus as a control and evaluated regarding their destruction. In assay B, eggs of E. paraensei were placed in Petri dishes with different supplemented culture media and with VC4 isolate and the destruction of eggs was examined at the end of 25 days of interaction. In assay C, effects of the crude extract of P. chlamydosporia (VC4) on eggs were evaluated at the end of 7 days. In assay A, there was no difference (p>0.05) in ovicidal activity among the tested isolates (VC1 and VC4); however, the highest percentage for ovicidal activity (type 3 effect) was demonstrated by the isolate VC4. In assay B, the culture medium starch-agar showed the best results for the destruction of the eggs, with a percentage of 46.6% at the end of the assay. In assay C, the crude extract of VC4 was effective in the destruction of E. paraensei eggs, with a percentage reduction of 53%. The results of this study demonstrate that a rich culture medium with a greater availability of carbon and nitrogen may interfere directly in the predatory characteristics of ovicidal fungi.

Efficacy of Clonostachys rosea and Duddingtonia flagrans in Reducing the Haemonchus contortus Infective Larvae

The biocontrol is proven effective in reducing in vitro and in situ free-living stages of major gastrointestinal helminths, allowing progress in reducing losses by parasitism, maximizing production, and productivity. This study aimed at evaluating the predatory activity of fungal isolates of Duddingtonia flagrans and Clonostachys rosea species and its association on infective larvae (L3) of H. contortus in microplots formed by grasses and maintained in a protected environment. All groups were added with 10 mL of an aqueous suspension with 618 H. contortus L3 approximately. Group 1 was used as control and only received the infective larvae. Groups 2 and 3 received D. flagrans chlamydospores and C. rosea conidia at doses of 5 × 106. Group 4 received the combination of 5 × 106 D. flagrans chlamydospores + 5 × 106 C. rosea conidia. D. flagrans and C. rosea showed nematicidal effectiveness reducing by 91.5 and 88.9%, respectively, the population of H. contortus L3. However, when used in combination efficiency decreased to 74.5% predation of H. contortus L3. These results demonstrate the need for further studies to determine the existence of additive effects, synergistic or antagonistic, between these species.

Amblyomma tick species infesting amphibians and reptiles in the seasonally dry Amazon forest, with new host records for Amblyomma rotundatum (Acari: Ixodida: Ixodidae)

Abstract Our study yielded a list of ticks found on amphibians and reptiles at five sites within seasonally dry Amazon forest in Mato Grosso State, central Brazil, in addition to new host records for A. rotundatum. We collected 431 tick specimens: 79 larvae, 115 nymphs, 38 females, and 199 males belonging to A. humerale, A. rotundatum, and Amblyomma sp., on 39 anurans and 21 reptiles. The toads R. guttatus and R. margaritifera and the frog L. pentadactylus are new hosts for A. rotundatum.

Nematophagous fungi combinations reduce free-living stages of sheep gastrointestinal nematodes in the field

Gastrointestinal nematodes (GIN) can reduce or limit sheep production. Currently there is a clear deficiency in the action of drugs for the control of these parasites. Nematophagous fungi are natural enemies of GIN. Fungal combinations have potential for reducing GIN populations. The aim of this study was to evaluate the efficiency combinations of nematophagous fungi in sodium alginate matrix pellets for the biological control agents of gastrointestinal sheep nematode parasites in the field. The nematophagous fungi (0.2mg of fungus per kg of body weight), Arthrobotrys conoides, A. robusta, Duddingtonia flagrans, and Monacrosporium thaumasium were used. The treated groups were administered mycelium combinations in the following combinations: group 1 (D. flagrans+A. robusta); group 2 (M. thaumasium+A. conoides). The control group did not receive any fungal pellets. We used three groups with eight Santa Inês sheep each. Each animal was treated with approximately 1g of pellet per 10kg of live weight. During the experimental period, we evaluated: number of eggs per gram of feces (EPG), infective larvae (L3) per kg of dry matter, larvae recovered from coprocultures, packed cell volume, total plasma protein concentration of sheep, and environmental conditions. Group 2 EPG (M. thaumasium+A. conoides) differed from the control group in September and October. The number of L3/kg of dry matter recovered from animals of groups 1 and 2 at distances of 0-20 and 20-40cm from the fecal pats was lower than the control group. The packed cell volume and total plasma proteins of treated animals were similar to those of the control group. The combination of treatment groups (D. flagrans+A. robusta and M. thaumasium+A. conoides) reduced the number of L3/kg of pasture. Therefore, treatment of nematophagous fungal combinations have the potential to manage free-living stages of GIN in sheep.

Culture medium characteristics on the predatory activity of the nematophagous fungus Duddingtonia flagrans

The influence of casein and pH on the activity of the nematophagous fungus Duddingtonia flagrans (AC001) on trichostrongylide larvae was evaluated. A ‘positive influence’ was observed contributing to the reduction of 63% in the average number of recovered L3 in the media supplemented with casein and pH 7.0.

Using the fungus Arthrobotrys cladodes var. macroides as a sustainable strategy to reduce numbers of infective larvae of bovine gastrointestinal parasitic nematodes

Research in the area of sanitation in ruminant production has focused on discovery of potential agents for biological control of helminths with nematophagous fungi and has provided evidence of success. The antagonistic potential of the fungus Arthrobotrys cladodes var. macroides on infective larvae of bovine gastrointestinal parasitic nematodes was evaluated by scanning electron microscopy. Additionally, an in vivo test of the resistance to digestive processes and viability of the fungus was carried out using a formulation based on sodium alginate administered orally in cattle. Production of conidia and chlamydospores was high. In in vitro tests, the number of infective nematode larvae was reduced 68.7% by the fungus in the treated group compared to the control group. The interaction between the fungus and the nematodes was confirmed by scanning electron microscopy. Plates containing fecal samples collected after oral administration of 100 g of pellets containing the A. cladodes fungus showed that the fungus survived passage through the gastrointestinal tract of ruminants, grew on agar, formed traps and preyed on L3 larvae of gastrointestinal parasites. The results of the present study provide a new opportunity for alternative, environmentally safe control of ruminant nematodes.

Reduction of bovine strongilides in naturally contaminated pastures in the southeast region of Brazil

Biological control through the use of nematophagous fungi is a sustainable alternative for combatting helminthes in domestic animals and allows a reduction in the use of anthelmintics. The objective of this research was to evaluate the efficacy of the Arthrobotrys cladodes var macroides fungus in a pelleted formulation, based on sodium alginate and administered twice a week orally, as an alternative for the biological control of nematodes in field-grown young cattle. The experiment was conducted in a farm located in the municipality of Viçosa, MG, where 12 cattle, seven to nine months old, were allocated in two groups (treated group and control group) and distributed in pickets of Brachiaria decumbens, naturally infested with nematode larvae. The animals in the treated group received 1g of sodium alginate matrix pellets for every 10 kg of animal live weight, containing the nematophagous fungus Arthrobotrys cladodes var macroides and administered twice a week in conjunction with commercial feed. In the control group, each animal received 1 g of pellets for every 10 kg of animal live weight, without fungal mycelium added to the feed. Samples of feces and pastures were collected fortnightly for 12 months. The results showed that the most prevalent nematode genera in the coprocultures were Haemonchus sp., Cooperia sp. and Oesophagostomum sp., reflecting the results found in forage. The pasture that contained the animals that received feed with the fungus presented a reduction of 59% and 52% of larvae recovered at distances of 20 cm and 40 cm from the fecal pats, respectively. The mean number of eggs per gram of feces each month and animal body weight did not differ (p > 0.05) between the treated and control groups. Stool and soil samples from both groups were colonized by A. cladodes fungus and other fungi. Administration of Arthrobotrys cladodes var macroides mycelium by means of a sodium alginate matrix twice weekly reduced larval infestation of the surrounding pasture, indicating that this fungus may be a promising biological control of infecting forms of nematodes present in the environment.