L3 Survival Research Articles

Comparative toxicity of coffee insecticides to the green lacewing Chrysoperla externa in laboratory and persistence trials

The green lacewing Chrysoperla externa (Hagen) (Neuroptera: Chrysopidae) is a generalist predator with a diet including major coffee pests. Aiming at a harmonious integration of biological and chemical control in coffee production systems, ecotoxicology studies are required to provide information on insecticide harmfulness toward natural enemies. We investigated the side effects of five coffee insecticides sprayed on eggs and third-instar larvae (L3) of C. externa. The assessed endpoints included C. externa viability (egg hatchability and L3 survival), developmental time, cycle completion, and body weight, sex ratio (female proportion) and accumulated oviposition of adults derived from the treated stages. Cyantraniliprole reduced viability of treated eggs, whereas abamectin prolonged the developmental time and reduced the oviposition of adults reared from the treated L3. In the F1 generation from treated L3, abamectin diminished egg viability and cycle completion and interfered with sex ratio and adult weight. Apart from these results, metaflumizone caused the most striking effects on C. externa by producing high L3 mortality (96%). The hazardousness of this compound, however, was dismissed in persistence bioassays (L3 exposure to aged residues in coffee leaves), indicating that metaflumizone requires direct spraying to kill C. externa. These findings allow us to conclude that while the other compounds (abamectin, abamectin + chlorantraniliprole, chlorantraniliprole and cyantraniliprole) are intrinsically safer for C. externa, metaflumizone applications should be performed in periods of low C. externa activity in plants to avoid direct predator exposure.

Larvicidal Efficacy of Ozone and Ultrasound on Angiostrongylus cantonensis (Rat Lungworm) Third-Stage Larvae.

The parasitic nematode Angiostrongylus cantonensis (rat lungworm) is the leading cause of human eosinophilic meningitis worldwide. Most human infections occur through the accidental consumption of A. cantonensis hidden within produce as infectious third-stage larvae (L3), yet little research has been published addressing possible methods to mitigate this means of transmission. Here, we describe our tests of ozone gas—an oxidizing agent—and ultrasound, both used for disinfection of food and municipal water supplies and in industrial cleaning. We found that exposure to ozone, produced using two different commercially available ozone generators over varying durations of time and concentrations, was capable of achieving 100% larval mortality. In addition, we evaluated the impact of different sound frequencies on A. cantonensis L3 survival using two different commercially available ultrasonic cleaners, and found that 60 s of 40 kHz produced 46% mortality within 2 h. The combined use of ultrasound and ozone gas simultaneously resulted in a minimum of 89% normalized mean percent mortality within 2 h of treatment. Our study suggests that both ozone and ultrasound show high larvicidal efficacy, both independently and together, and thus show promise as methods for reducing the risk of rat lungworm infection via accidental consumption.

High levels of third-stage larvae (L3) overwinter survival for multiple cattle gastrointestinal nematode species on western Canadian pastures as revealed by ITS2 rDNA metabarcoding

BackgroundThe ability of infective larvae of cattle gastrointestinal nematode (GIN) species to overwinter on pastures in northerly climatic zones with very cold dry winters is poorly understood. This is an important knowledge gap with critical implications for parasite risk assessment and control.MethodsInfective third-stage larvae (L3) were quantified in samples of fecal pats, together with adjacent grass and soil, before and after winter on three farms in southern, central and northern Alberta. Nemabiome ITS2 metabarcoding was then performed on the harvested L3 populations to determine the species composition. Finally, parasite-free tracer calves were used to investigate if the L3 surviving the winter could infect calves and develop to adult worms in spring.ResultsFarm level monitoring, using solar powered weather stations, revealed that ground temperatures were consistently higher, and less variable, than the air temperatures; minimum winter air and ground temperatures were − 32.5 °C and − 24.7 °C respectively. In spite of the extremely low minimum temperatures reached, L3 were recovered from fecal pats and grass before and after winter with only a 38% and 61% overall reduction over the winter, respectively. Nemabiome ITS2 metabarcoding assay revealed that the proportion of L3 surviving the winter was high for both Cooperia oncophora and Ostertagia ostertagi although survival of the former species was statistically significantly higher than the latter. Nematodirus helvetinaus and Trichostrongylus axei could be detected after winter whereas Haemonchus placei L3 could not overwinter at all. Adult C. oncophora, O. ostertagi and N. helvetianus could be recovered from tracer calves grazing after the winter.ConclusionsThe largest proportion of L3 were recovered from fecal pats suggesting this is important refuge for L3 survival. Results also show that L3 of several GIN parasite species can survive relatively efficiently on pastures even in the extreme winter conditions in western Canada. Tracer calf experiments confirmed that overwintered L3 of both C. oncophora and O. ostertagi were capable of establishing a patent infection in the following spring. These results have important implications for the epidemiology, risk of production impact and the design of effective control strategies. The work also illustrates the value of applying ITS2 nemabiome metabarcoding to environmental samples.

A model for the dynamics of the free-living stages of equine cyathostomins

Anthelmintic resistance in strongyle nematode parasites of horses is an expanding global problem and steps need to be taken to slow its development before control becomes more problematic. A move away from traditional deworming programmes, involving frequent whole-herd treatments with broad spectrum anthelmintics, to a more strategic or targeted use of chemicals is required. However, anthelmintic resistance management strategies which also maintain effective control are invariably more complicated and often require a greater understanding of both nematode epidemiology and grazing management, than does the simple routine use of chemicals. Here, as a first step in applying a modelling approach to resistance management in horses, a model is proposed to describe the dynamics on pasture of the free-living stages of equine cyathostomins. Firstly, the development and survival of the pre-infective stages is considered as a single process driven by temperature, and secondly, two populations of infective stage larvae (L3) are considered; those within the faecal pat and those on the herbage. Both are modelled using the box-car train approach which allows for variable development rates within a cohort of individuals and full overlap of generations. Uniquely, L3 survival is modelled as an ageing process where larvae progress through physiological age classes at a rate determined by temperature and rainfall. Model output reflects the dynamics of free-living stages under a range of environments. Under extreme cold, there is no development to L3 but eggs can survive for long periods to develop if conditions become favourable, while L3 survival is reduced under repeated freeze–thaw cycles. Under tropical conditions, development is rapid and a large number of L3 can be produced but survival of L3 is short. In temperate climates development tends to be slower, with large numbers of L3 produced over the warmer months but fewer over winter, and L3 survival tends to be higher all year round. Although attempts to validate model output against field studies were compromised by the lack of published detail or an inability to access specific weather records, outputs averaged over multiple sets of weather data was often appropriate for that location. Variation in model output when using weather data sets which started on different days within the same week suggests that day-to-day differences in weather may affect the number of L3 developing on pasture and that optimisation of anthelmintic use to minimise pasture infectivity may require a more detailed understanding of weather effects than previously thought.

Desiccation tolerance of gastrointestinal nematode third-stage larvae: exploring the effects on survival and fitness.

The free-living third-stage larvae (L3) of gastrointestinal nematodes are able to tolerate extreme weather conditions such as desiccation, but little is known about the consequent effects this has on their fitness. This study explored how the desiccation of Haemonchus contortus L3 larvae affected their absolute fitness by examining their success at consequent life cycle stages for a complete generation, and comparing them against a control. The stages examined include establishment, fecundity, larval development and pathogenicity. The results show that while desiccation greatly reduced the survival of the L3 prior to infection in sheep, their absolute fitness was not negatively impacted. Instead, it appears desiccation slightly augmented H. contortus fitness by triggering increases in fecundity. The study further explored what influence different gastrointestinal nematode (GIN) species (H. contortus, Trichostrongylus colubriformis, Teladorsagia circumcincta), isolates and age of L3 had on their capacity to revive following various periods of desiccation. The results showed desiccation tolerance varied as a function of each of these variables. The greatest L3 survival was found in Te. circumcincta followed by Tr. colubriformis and finally H. contortus. Significant variation was observed between individual species isolates and as a function of age. The results of this study carry important practical implications for the epidemiological understanding of gastrointestinal nematode species of economic importance.

Observations on the free-living stages of cattle gastrointestinal nematodes

A 4-year study on the free-living stages of cattle gastrointestinal nematodes was conducted to determine (a) the development time from egg to infective larvae (L3) inside the faecal pats, (b) the pasture infectivity levels over time, and (c) the survival of L3 on pasture. Naturally infected calves were allowed to contaminate 16 plots on monthly basis. Weekly monitoring of eggs per gram of faeces (epg) values and faecal cultures from these animals provided data for the contamination patterns and the relative nematode population composition. At the same time, faecal pats were shaped and deposited monthly onto herbage and sampled weekly to determine the development time from egg to L3.Herbage samples were collected fortnightly over a 16-month period after deposition to evaluate the pasture larval infectivity and survival of L3 over time. The development time from egg to L3 was 1–2 weeks in summer, 3–5 weeks in autumn, 4–6 weeks in winter, and 1–4 weeks in spring. The levels of contamination and pasture infectivity showed a clear seasonality during autumn–winter and spring, whilst a high mortality of larvae on pasture occurred in summer. Ostertagia spp., Cooperia spp. and Trichostrongylus spp. were predominant and a survival of L3 on pasture over a 1-year period was recorded in this study.

Sensitization with anti-inflammatory BmAFI of Brugia malayi allows L3 development in the hostile peritoneal cavity of Mastomys coucha

Filarial parasites survive by inducing tolerance in host but the antigens and mechanisms involved are not clear. Recently we found that BmAFI, a Sephadex G-200 eluted fraction of Brugia malayi adult worm extract, stimulates IL-10 release from THP-1 cells. In the present study, we determined the SDS-PAGE profile of BmAFI and infective 3rd stage larva (L3), investigated the effect of pre-sensitization of host with BmAFI on the survival and development of L3 in the non-permissive peritoneal cavity (p.c.) of the permissive host Mastomys coucha and in the p.c. of non-permissive Swiss mice, and studied immunological correlates for the observed effects. The parasite development and burden in p.c., was determined in sensitized infected M. coucha and Swiss mice and the release of TGF-β, IL-4, IL-10, IL-13, IFN-γ and NO, cellular proliferative response to Con A and BmAFI and levels of IgG subclasses and IgE were determined in sensitized infected M. coucha. Cellular proliferative response to Con A and BmAFI, mRNA expression of GATA-3, CTLA-4 and T-bet were determined in sensitized Swiss mice. In addition, the parasitological parameter was also studied in BmAFI-sensitized M. coucha exposed to the infection by standard subcutaneous (s.c.) route to assess whether sensitization enhances the intensity of infection. BmAFI-sensitization permitted survival of L3 and their development to adult stage by day 60 p.i. in the p.c. of M. coucha; in non-sensitized animals L3 could molt to L4 only and no parasite could be recovered beyond day 30 p.i. In M. coucha that received infection by s.c. route, pre-sensitization with BmAFI enhanced the microfilaraemia and adult worm recovery. In sensitized Swiss mice L3 could successfully molt to L4 in p.c. with improved recovery of parasite. BmAFI sensitization upregulated TGF-β and IL-10 release, IgG1 and IgG2b levels, GATA-3 and CTLA-4 mRNA expression, suppressed the cellular proliferative response and downregulated Con A stimulated response, IgE, IL-13, IFN-γ and NO responses. Immunoblot analysis showed that the BmAFI antiserum also strongly reacts with some L3 molecules. The results show, for the first time, that sensitization with the anti-inflammatory BmAFI which shares some of its molecules with those in L3, facilitates parasite survival in the non-permissive p.c. of the permissive host M. coucha, render a non-permissive Swiss mouse partially permissive to infection and enhances parasite load in M. coucha receiving the infection through permissive s.c. route by evoking a modified Th2 type of response and anti-inflammatory milieu. In conclusion, the findings suggest that the anti-inflammatory BmAFI fraction facilitates survival of B. malayi infection even in non-permissive environment.

Vertical migration of Haemonchus contortus infective larvae on Cynodon dactylon and Paspalum notatum pastures in response to climatic conditions

Observations were made on vertical migration patterns of Haemonchus contortus infective larvae on Cynodon dactylon (bermudagrass) and Paspalum notatum (bahiagrass) pastures under summer climatic conditions typical of East Texas. Ten thousand H. contortus infective larvae (L3) were introduced to 100 cm 2 subplots of each pasture species within a plot area of 1 m 2. Subplots were inoculated with larvae by applying them in an aqueous medium to the soil or mat beneath the vegetation. Herbage from the inoculated areas was harvested on 5 sampling days over a span of 21 days. L3 recoveries were observed and recorded each day on four herbage strata viz. 0–5, 5–10, 10–20 and >20 cm from ground level. The log transformed larval recovery data were analyzed for effect of day, stratum, and day × stratum interaction for each grass species during two separate experimental periods. Precipitation, relative humidity and temperature during the study were subjected to correlation and multiple regression analyses with the larval counts. Significant ( P ≤ 0.0017) differences were found for the effect of day on larval recoveries. No significant differences were detected for stratum or day × stratum interaction effects, though stratum provided a strong indication of influencing larval recovery. A high positive correlation (≥0.93) between rainfall and total average daily larval counts was apparent. The multiple regression analysis did not show significant results for any of the climatic factors tested. This study showed that the H. contortus infective larvae can survive beyond 21 days in the soil and infest pasture grasses when the climatic conditions are favorable. Avoiding use of H. contortus contaminated pasturelands in summer at the onset of rainfall following a dry spell may effectively reduce nematode loads in susceptible farm animals. Additional studies should focus on factors affecting long term L3 survivability, migrational pattern on these and other plant species and the relationship between climatic factors and larval migration patterns throughout the year. Total larval recovery of H. contortus in this study was greater in bahiagrass than bermudagrass. While the design of this study did not allow for testing one pasture species against another, studies with potted plants would allow for some valid comparisons. Soil characteristics may also play a role in L3 survival and subsequent migration.

Desenvolvimento e migraÇão de larvas infectantes de ciatostomíneos (Nematoda: Cyathostominae) em gramínea coast cross (Cynodon dactylon) em clima tropical, na Baixada Fluminense, RJ, Brasil

A study following the development and migration of Cyathostominae infective larvae was conducted from July 2003 to November 2004 in tropical climate, Baixada Fluminense, RJ, Brazil. Samples of naturally infected feces were placed on 12 m2 plot each month on a cyathostomin-free "Bermuda grass" pasture (Cynodon dactylon). After Seven days, samples of feces and grass were collected every week at 8 a.m, 1 and 5 p.m., weighed and processed by Baermann technique. Higher survival of L3 was found at dry season, 15 and 12 weeks on feces and sward respectively, at rainy season the survival was smaller. The multivariable analysis of main components was evident the influence of time and environment variables on L3 recovery from feces and grass. Close relationship between time and the number of L3 in feces could be noted, in contrast with L3 in sward. The climatic conditions influenced directly the number of infective larvae. The infective larvae were recovered during three times and the Kruskal-Wallis test did not present significance among them.

Antibacterial and Antiparasitic Activity of Oleanolic Acid and its Glycosides isolated from Marigold (Calendula officinalis)

The antibacterial and antiparasitic activities of free oleanolic acid and its glucosides and glucuronides isolated from marigold (Calendula officinalis) were investigated. The MIC of oleanolic acid and the effect on bacterial growth were estimated by A600 measurements. Oleanolic acid's influence on bacterial survival and the ability to induce autolysis were measured by counting the number of cfu. Cell morphology and the presence of endospores were observed under electron and light microscopy, respectively. Oleanolic acid inhibited bacterial growth and survival, influenced cell morphology and enhanced the autolysis of Gram-positive bacteria suggesting that bacterial envelopes are the target of its activity. On the other hand, glycosides of oleanolic acid inhibited the development of L3 Heligmosomoides polygyrus larvae, the infective stage of this intestinal parasitic nematode. In addition, both oleanolic acid and its glycosides reduced the rate of L3 survival during prolonged storage, but only oleanolic acid glucuronides affected nematode infectivity. The presented results suggest that oleanolic acid and its glycosides can be considered as potential therapeutic agents.

Cryopreservation of Infective Larvae of Onchocerca volvulus (Filarioidea: Onchocercidae)

Infective larvae (L3) of Onchocerca volvulus were procured in Liberia, West Africa, in the natural black fly vector, Simulium yahense. A cryobiological technique was developed to preserve L3 of O. volvulus that were fully viable after thawing. Larvae were treated before cooling with 4 cryoprotective compounds. Three compounds, dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol, were prepared with distilled water. The fourth compound was DMSO prepared in different concentrations with 0.25 M sucrose. The treatment with DMSO + 0.25 M sucrose cryoprotectant resulted in the highest survival of infective larvae. Five cooling rates between 0.5 C/min and 20.0 C/min were applied. The highest survival of L3 was with the cooling rate of 1.0 C/min. Two-step cooling of L3 was applied. In the first step, L3's were frozen to 5 levels from -10.0 C to -20.0 C, -30.0 C, -40.0 C, -60 C, and -80.0 C, and in the second step, larvae were transferred into liquid nitrogen at -196 C for rapid cooling and storage. The survival was the highest when larvae were cooled to approximately -40 C prior to transfer into liquid nitrogen. Slow, gradual, and rapid thawing procedures were applied. The survival was the highest in rapid warming.

Cryopreservation of Angiostrongylus cantonensis third-stage larvae

An optimum protocol for the cryopreservation of third-stage larvae (L3) of Angiostrongylus cantonensis was determined by: (i) use of different dimethyl sulphoxide (DMSO) concentrations; (ii) use of different FBS concentrations in RPMI-1640 medium; and (iii) variation in the thawing temperature. Studies showed that with concentrations of DMSO at 4.8% and FBS at 40% and a thawing temperature of 43 degrees C, L3 survival assessed by motility was highest at 56%. The infectivity of the cryopreserved L3 of A. cantonensis was 20% compared to controls obtained from slugs.

Ecology and Control of Gastrointestinal Nematodes of Beef Cattle

A 4-year study on the free-living stages of cattle gastrointestinal nematodes was conducted to determine (a) the development time from egg to infective larvae (L3) inside the faecal pats, (b) the pasture infectivity levels over time, and (c) the survival of L3 on pasture. Naturally infected calves were allowed to contaminate 16 plots on monthly basis. Weekly monitoring of eggs per gram of faeces (epg) values and faecal cultures from these animals provided data for the contamination patterns and the relative nematode population composition. At the same time, faecal pats were shaped and deposited monthly onto herbage and sampled weekly to determine the development time from egg to L3.Herbage samples were collected fortnightly over a 16-month period after deposition to evaluate the pasture larval infectivity and survival of L3 over time. The development time from egg to L3 was 1–2 weeks in summer, 3–5 weeks in autumn, 4–6 weeks in winter, and 1–4 weeks in spring. The levels of contamination and pasture infectivity showed a clear seasonality during autumn–winter and spring, whilst a high mortality of larvae on pasture occurred in summer. Ostertagia spp., Cooperia spp. and Trichostrongylus spp. were predominant and a survival of L3 on pasture over a 1-year period was recorded in this study.