Identification of the intermediate host of Gongylonema sp., the etiological agent of the necrotic oropharyngeal disease of the Scops owl (Otus scops)

Since 1997, fledgling Scops owls (Chordata: Strigidae) have been brought to the Brinzal Owl Rescue Centre (Madrid, Spain) with severe lesions in their oral cavities. Lesions consist of the presence of proliferative necrotic material in the oral cavity resulting in white plaques, which can lead to death by starvation. This disease has been detected in owls only within the limits of the city of Madrid. The etiologic agent has been identified as Gongylonema sp. (Nematoda: Spirurida), a nematode genus that includes a coprophagous arthropod as intermediate host in its cycle. The aim of this study was to identify the intermediate host of the parasite. Our work was structured in four component phases: i) Diet study of newborn chicks; ii) trapping arthropods that could be intermediate hosts; iii) molecular detection of the parasite in the selected arthropods: and iv) molecular characterization of the detected parasites by amplifying the cox1 gene. Four male owls were radio-tagged in order to locate their nests and a camera trap was placed to identify the prey brought to the owlets. Secondly, the arthropods present in the hunting areas of the owls were sampled, identified and analyzed by real time PCR (rtPCR). Only oriental cockroaches, B. orientalis (Arthropoda: Blattodea), were positive by rtPCR detection of Gongylonema sp. (66.7%). The nematodes obtained from cockroaches had a 99.8% identity of the cox1 gene with the Gongylonema sp. isolated for the first time in a Scops owl. Furthermore, these sequences only showed an <89% identity with all the other Gongylonema sequences available in the GenBank database. We conclude that the oriental cockroach should be considered as an intermediate host of the etiologic agent of NOD.     

Disease prevalence in an urban raptor related to pest species: The case of Eurasian Scops Owl Otus scops infection by Gongylonema sp.

Urbanization is often associated with homogenization, including the homogenization of biodiversity and overpopulation by generalist species that can change community and disease dynamics. In Madrid, nestlings of Eurasian Scops Owl Otus scops frequently suffer necrotic oropharyngeal disease as a result of infection by Gongylonema sp., a parasitic nematode transmitted by adult owls to their offspring through the diet, more specifically through consumption of a pest species: the oriental cockroach Blatta orientalis. We studied how the presence of cockroaches and owl infection rates are affected by urban features such as green areas. We found that a higher number of birds were affected by the parasite in the initial period of the breeding season and in areas with greater surface area of green spaces. We suggest that urban management should promote the diversity of insects in green areas so that the owls diversify their dietary offer to their offspring, to reduce the prevalence of the disease and improve their breeding success in the city of Madrid, as this problem has not yet been described in other cities.     

Natural parasitism of Romanomermis iyengari (Welch) (Nematoda: Mermithidae) on various species of mosquitoes breeding in rice fields in Pondicherry,India

Natural habitat of Romanomermis iyengari, an entomophilic mermithid nematode, is rice field. Parasitism of this nematode on mosquito larvae breeding in rice fields in Pondicherry, India, has been studied in 1979. The present study was to find out its persistence in the same habitat, after many years of agricultural practices. Evaluation of parasitism on mosquito larvae was carried out during two rice cultivation seasons at a schedule of 1 week prior, and 1 and 2 weeks after transplantation of paddy. Larval stages of mosquito species such as Culex tritaeniorhynchus, Culex vishnui, Anopheles vagus, and Anopheles subpictus showed parasitism of R. iyengari. Parasitism was the highest on C. tritaeniorhynchus with a maximum of 15.38% during season I and 17.85% during season II. Followed by this, it was on A. vagus (10.82% and 9.43%), C. vishnui (7.37% and 8.69%), and A. subpictus (3.70% and 6.36%). The overall density of mosquito larvae and level of parasitism of R. iyengari on them during season II was significantly higher than that of season I. The parasitism was maximum at 1 week before transplantation and showed a decreasing trend towards 2 weeks after transplantation, during both the seasons. The level of natural parasitism of R. iyengari during the present investigation was higher than that observed in the same ecosystem almost 35 years ago. This is clearly an indication towards the capability of the nematode to withstand several years of agricultural practices, including pesticide and agrichemical application. Hence, if applied at sufficient level and frequency, this nematode could act as a successful biocontrol agent against mosquitoes breeding in rice fields.     

Modifying orchard planting sites conserves entomopathogenic nematodes,reduces weevil herbivory and increases citrus tree growth,survival and fruit yield

In Florida, a root weevil pest of citrus, Diaprepes abbreviatus, is more damaging and attains higher population density in some orchards on fine textured, poorly drained “flatwoods” soils than in those on the deep, coarse sandy soils of the central ridge. Previous research revealed that sentinel weevil larvae were killed by indigenous entomopathogenic nematodes (EPNs) at significantly higher rates in an orchard on the central ridge, compared to one in the flatwoods. We hypothesized that filling tree planting holes in a flatwoods orchard with sandy soil from the central ridge would provide a more suitable habitat for EPNs, thereby reducing weevil numbers and root herbivory. Fifty trees were planted in oversized planting holes filled with coarse sand and 50 trees were planted in native soil in a split plot design where whole plots were species of introduced EPNs and split plots were soil type. Each of Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, or no EPNs were introduced into the rhizospheres in 10 plots of each soil type. During four years, EPN numbers in soil samples and the relative abundance of seven species of nematophagous fungi associated with nematodes were measured three times using real-time PCR. The efficacy of EPNs against sentinel weevil larvae was also measured three times by burying caged weevils in situ. EPN species richness (P = 0.001) and diversity (P = 0.01) were always higher in sand than native soil. Soil type had no effect on numbers of EPNs in samples, but EPNs were detected more frequently (P = 0.01) in plots of sandy soil than native soil in 2011. Two nematophagous fungi species, Paecilomyces lilacinus and Catenaria sp. were significantly more abundant in nematode samples from sandy soil on all three sampling dates. Efficacy of EPNs against weevil larvae was greater in sandy soil inoculated with S. diaprepesi (P = 0.03) in June 2010 and in all treatments in sandy soil in May 2011 (P = 0.03). Sixty-eight percent more adult weevils (P = 0.01) were trapped emerging from native soil during two years than from sandy soil. By May 2011, the cumulative number of weevils emerging from each plot was inversely related (P = 0.01) to the numbers of EPNs detected in plots and to EPN efficacy against sentinels. Three trees in sandy soil died as a result of root herbivory compared to 21 trees in native soil. Surviving trees in sandy soil had trunk diameters that were 60% larger (P = 0.001) and produced 85% more fruit (P = 0.001) than those in native soil. Although it is not possible to characterize all of the mechanisms by which the two soil treatments affected weevils and trees, substitution of sand for native soil was an effective means of conserving EPNs and shows promise as a cultural practice to manage D. abbreviatus in flatwoods citrus orchards with a history of weevil damage to trees.     

Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification

Trichinella spiralis is a tissue-dwelling nematode parasite. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the sensitive and rapid detection of T. spiralis larvae in muscle samples. Sixteen sets of primers were designed to recognise distinct sequences of a conserved gene, a 1.6 kb repetitive element of the Trichinella genome. One set of primers was selected as the most appropriate for rapid detection. The specificity and sensitivity of the primers in LAMP reactions for T. spiralis larvae and muscle samples of mice infected with T. spiralis were determined. Another 10 heterologous parasites were selected for specificity assays. The results showed that target DNA was amplified and visualised by monitoring turbidity and adding calcein detection methods within 70 min at an isothermal temperature of 63 °C. The sensitivity of LAMP with the detection limit of 362 fg/μl was >10 times higher than that for PCR. The designed primers had a good specificity. No cross-reactivity was found with the DNA of any other parasites. The assay was able to detect T. spiralis in all mouse muscle samples infected with 10 T. spiralis larvae on day 20 p.i. We believe this is the first report regarding the application of the LAMP assay for detection of T. spiralis larvae in muscle samples from experimentally infected mice. This method demonstrates a potentially valuable means for the direct detection of T. spiralis larvae in meat inspection.     

Long-term effects and persistence of Steinernema scarabaei applied for suppression of Anomala orientalis (Coleoptera: Scarabaeidae)

The entomopathogenic nematode, Steinernema scarabaei, is adapted to scarab larvae as hosts and has already shown exceptional potential for inundative control of these pests. To determine the long-term effects of S. scarabaei application on scarab populations and the nematode’s persistence, S. scarabaei was applied in mid-September at rates from 0.06 to 2.5 × 10⁹ infective juveniles (IJs)/ha to turfgrass plots seeded with oriental beetle, Anomala orientalis, larvae. Scarab and nematode populations were monitored for 3–4 years thereafter. S. scarabaei provided excellent A. orientalis control (77–100%) within 1 month of application at rates of 0.25–2.5 × 10⁹ (IJs)/ha and particularly in the following spring at rates of 0.1–2.5 × 10⁹ (IJs)/ha (86–100%). S. scarabaei provided significant control in the next A. orientalis generation in two out of 10 treatments in fall (i.e., 13 months after application) and six out of 10 treatments in the following spring. Thereafter, significant control was only observed occasionally. S. scarabaei numbers were highly variable, and few significant differences among treatments were observed. S. scarabaei recovery from the treated plots was generally more consistent through the first spring after application and became more variable thereafter, but S. scarabaei was recovered for up to 4 years in the experimental plots. Endemic populations of Heterorhabditis bacteriophora and Steinernema carpocapsae, regularly recovered from the experimental plots and often in higher numbers than S. scarabaei, had no significant effect on A. orientalis densities but were able to coexist with S. scarabaei. Our observations suggest that, once current problems with its mass production can be overcome, S. scarabaei could be augmented periodically in areas with recurrent scarab infestations to provide long-term suppression.     

Distribution of nematode larvae of sheep in tropical pasture plants

The aim of this work was to study the dynamics of parasitic nematode larvae of sheep (third larval stage), in tropical forage species. The experiment was composed of two different dry matter yield for each plant species, Pensacola grass (Paspalum saurae) and Aruana grass (Panicum maximum). The animals in the experiment were 28 Suffolk lambs that were 6–8 months old. Lambs were left in a naturally contaminated pasture for 86 days. A randomized design was adopted, collection of pasture was made every 15 days, separated into upper and lower portions and made larval enumeration. Lambs were evaluated by faecal egg count (FEC) to monitoring worm infection. The number of parasite larvae in both forages was similar (p > 0.05). However, higher (p < 0.05) infestation by helminth larvae in forage with lower dry matter yield, was observed in the upper portion of both plants studied. Animals with lower forage yield, for both forages, presented superior averages (p < 0.05) of FEC compared to higher forage yield pasture. Lambs grazing on Pensacola grass, with lower dry matter yield, showed increasing FECs over time. Lambs maintained on the pasture with higher yield of dry matter (Aruana) showed decreasing FECs over time. Similar results were observed when each pasture type was analysed for larval contamination. Epidemiologic and management implications are discussed in this work.     

Ecological analysis of nematodes associated with vegetable-maize cropping system at Dhapa,Kolkata (India)

The study focused on the ecological analysis of plant and soil nematode communities associated with vegetables-maize crop sequences in the eastern fringe of Kolkata at Dhapa from December 2011 to April 2014 at a monthly interval. Plant-parasitic nematodes like Pratylenchus zeae, Meloidogyne incognita, Hoplolaimus indicus, Helicotylenchus dihystera, Tylenchorhynchus brevilineatus, Rotylenchulus reniformis, and Criconemoides onoensis were observed in two major cropping sequences of chilli-amaranthus-maize-radish-cauliflower and amaranthus-maize-cabbage-chilli. Among them, R. reniformis was found to be the most prevalent genera with the highest relative prominence value (RPV) of 27.29 followed by P. zeae (20.30) and T. brevilineatus (16.70). The population dynamics of P. zeae revealed the lowest density of the nematode was recorded (26 per 200 cc of soil + 5 g of the root) in March and the highest (634) in September. The variation in the abundances of other PPNs was also noted, and that attributed to the crops in the crop sequence and weather conditions. Low numbers of R. reniformis was found in the maize rhizosphere. The saprozoic nematodes (SNs) maintained higher mean densities (1734/200 cc soil); they were recorded in high numbers in the rainy season as compared to other seasons. No significant differences in the Shannon diversity index (H′), evenness (J) were observed across the growing period. A significant relationship was found between nematode abundance and climatic variables observed. This study provides baseline information on nematodes associated with maize-based cropping system, population abundance, Mylonchulus-SNs relationships, and population fluctuation in response to environmental factors.     

Interaction between Meloidogyne incognita and Rhizoctonia solani on green beans

The interaction between Meloidogyne incognita (race 2) and Rhizoctonia solani (AG 4) in a root rot disease complex of green beans (Phaseolus vulgaris) was examined in a greenhouse pot experiment. Three week-old seedlings (cv. Contender) were inoculated with the nematode and/or the fungus in different combinations and sequences. Two months after last nematode inoculation, the test was terminated and data were recorded. The synchronized inoculation by both pathogens (N + F) increased the index of Rhizoctonia root rot and the number of root galls; and suppressed plant growth, compared to controls. However, the severity of root rot and suppression of plant growth were greater and more evident when inoculation by the nematode preceded the fungus (N → F) by two weeks. Nematode reproduction (eggs/g root) was adversely affected by the presence of the fungus except by the synchronized inoculation. When inoculation by nematode preceded the fungus, plant growth was severely suppressed and roots were highly damaged and rotted leading to a decrease of root galls and eggs.     

Heligmosomoides polygyrus: EAE remission is correlated with different systemic cytokine profiles provoked by L4 and adult nematodes

Primary exposure of mice to gastrointestinal nematode infection with Heligmosomoides polygyrus reduces inflammation in an experimental model of multiple sclerosis. In this study, we aimed to evaluate the ability of H. polygyrus L4 larvae and adults infection to reduce the symptoms of ongoing experimental autoimmune encephalomyelitis (EAE) in female C57Bl/6 mice. EAE was induced by myelin oligodendrocyte glycoprotein MOG_p35–55 and after 21 days mice were orally infected with 200 infective larvae (L3) of H. polygyrus. Reduction in EAE symptoms was observed from 2 days post infection and the symptoms were almost completely inhibited at 6 days post infection. This effect was associated with limited total protein content in the cerebrospinal fluid; CSF, and significant decreased pro-inflammatory IL-12p40 concentration and increased concentration of the regulatory cytokines IL-10, TGF-β and IL-6 in the CSF and in the serum. The reduction of EAE symptoms in the enteral phase was associated with higher IL-12p40 concentration in the CSF and very low concentrations of IL-17A and IL-2 in the serum. The fourth stage of gastrointestinal nematode can reverse systemic inflammation in animal models of multiple sclerosis by reducing IL-12 and promoting regulatory cytokines production. The mechanism induced by adult nematodes which sustained EAE inhibition can be provoked by regulatory mechanism connected with reduce IL-17A concentration.     

Anisakis spp. larvae in three mesopelagic and bathypelagic fish species of the central Mediterranean Sea

In this work 437 fish samples of species belonging to the families Myctophidae (Electrona risso and Diaphus metopoclampus) and Phosichthyidae (Vinciguerria attenuata) were examined for the presence of Anisakidae larvae. The study was performed with fishes in the central Mediterranean Sea, particularly in the Strait of Sicily and in the Strait of Messina. The visual inspection and chloro-peptic analysis revealed the presence of nematode parasites with prevalence values between 2.9% in Electrona risso samples and 5.4% in Vinciguerria attenuata samples. A positive correlation was found between standard length (SL) and prevalence of infestation in D. metopoclampus samples (p < 0.05). The larvae examined were morphologically ascribed, at genus level, to Anisakis morphotypes I and II and molecularly identified as Anisakis pegreffii, Anisakis ziphidarum and Anisakis physeteris, in 67%, 9% and 24% of the fish samples examined. Overall, A. pegreffii and A. ziphidarum larvae were isolated in 14 and 2 specimens of D. metopoclampus respectively, A. physeteris larvae were found in 3 E. risso and 2 V. attenuata. A positive correlation was found between standard length and prevalence of infestation in D. metopoclampus samples (p < 0.05). First information is provided on the presence of Anisakis spp. larvae of the myctophid fish species E. risso, D. metopoclampus and V. attenuata from the Central Mediterranean. It is also confirmed the role of lanternfishes (Myctophidae) as paratenic hosts for Anisakis spp.     

Bioassays for comparative infectivity of mermithid nematodes (Romanomermis iyengari,Romanomermis culicivorax and Strelkovimermis spiculatus) for culicine mosquito larvae

Two improved bioassays were developed to establish infectivity baselines for selection experiments using mermithid nematode variants. Comparative infectivity of Romanomermis iyengari, Romanomermis culicivorax and Strelkovimermis spiculatus using larvae of three mosquito spp. Aedes sierrensis, Aedes aegypti and Culex pipiens were evaluated with “plate” and “tray” bioassays at selected intensity of infections. Using the “plate” bioassay, single mosquito larvae were immersed in 2 ml of water within individual depressions of 12-well, polystyrene tissue culture plates. One, three, or five preparasitic juveniles (J2) were added to each well. In the “tray” bioassay, polyethylene trays containing 500 ml water and 100 mosquito larvae were exposed to 500 (5:1, nematode:insect host) or 1000 (10:1) J2s. Percentage infection (PINF, infectivity) and intensity of infection (IINF, #nematodes per infected larvae) number were determined only after emergence of post-parasitic J3 juveniles. Under the bioassay conditions, all three species of nematodes resulted in infections in all mosquito hosts, but R. iyengari exhibited better effectiveness in the parasitism of mosquito larvae. The three species of mosquitoes presented high levels of susceptibility to each of the three species of nematodes, but in general Cx. pipiens and Ae. sierrensis were slightly more susceptible than Ae. aegypti. The “plate” bioassay was more efficient in measurement of infectivity of the mermithid species and in establishing baseline characteristics for these mosquito-parasitic nematodes. The “tray” bioassay was an effective bioassay for large cohorts of both infective juveniles and host larvae and, potential for field interactions.     

Interannual patterns of the large free-living nematode assemblages in the Mexican Exclusive Economic Zone,NW Gulf of Mexico after the Deepwater Horizon oil spill

The presence and abundance of particular nematode genera in marine sediments are bioindicators of environmental disturbance. They can even reveal the existence of specific toxicants, such as trace-metals and hydrocarbons derived from fossil fuels. We studied this important infaunal component during a three-year monitoring program in Mexico’s Exclusive Economic Zone (EEZ) in the aftermath of a major oil spill in the northern Gulf of Mexico caused by the sinking of the Deepwater Horizon platform (DWH) in April of 2010. A closer look at the nematode composition and density values throughout and after the DWH oil spill revealed considerable changes. Continental shelf and upper slope sediments sampled in the summer of 2010 included 48 genera and a density of 44.45 ind/10 cm⁻². Eight months later in the winter of 2011, there was a critical decrement in genera number (23) and density (25.22 ind/10 cm⁻²). However, in 2012, the nematode community showed signs of recovery: genus diversity was 58 with a density of 91.45 ind/10 cm⁻². Significant differences were recorded between the first and the last sampling periods, driven by a remarkable turn-over in genera composition, and by an increase in density of the genera Sabatieria, Dorylaimopsis, and Cheironchus. Similarly, significant spatial differences between the nematofauna of the inner-middle shelf and the deepest zone (>2000 m) were detected. Correlation analyses confirmed the tolerant behavior of the above genera to Ni and Co, and their proliferation when hydrocarbon compounds presumably derived from the DWH oil spill increased throughout the study. The Maturity Index (MI) estimated for each survey ranged from 2.35 to 2.6. Environmental Quality Status conditions in the summer of 2010 were good while in the winter of 2011 were poor, and moderate towards the winter of 2012. Index of Trophic Diversity (ITD) values for each survey were high: 0.70, 0.68, and 0.74, respectively. A high predominance of feeding guilds 1B and 2B was recorded throughout the study.The estimated Index of Trophic Diversity (ITD) values resulted high (Table 5). These values apparently indicated that the four feeding types were proportionally distributed in the nematode assemblage. This fact was better reflected during the winter of 2012. Nonetheless, these results are not congruent with the high predominance of feeding guilds 1B and 2B observed throughout the study.     

Assessment of frozen larvae of Callosobruchus maculatus as hosts for rearing Pteromalus cerealellae (Ashmead) (Hymenoptera: Pteromalidae)

The suitability of frozen host larvae for rearing Pteromalus cerealellae (Ashmead) (Hymenoptera: Pteromalidae), an ectoparasitoid of Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae) and other stored-product insects was investigated. The reproductive potential (number and sex ratio of progeny) of female P. cerealellae was compared on live (fresh) C. maculatus larvae (concealed within cowpea seeds) versus frozen larvae (obtained by freezing infested cowpea seeds at ?20 °C for 48 h) which were subsequently thawed and held at ambient conditions (～25 ± 1 °C, 50 ± 5% RH) for 4, 24, 48, 72, 96, and 120 h before exposure to female parasitoids. No significant differences were recorded in the numbers and sex ratios of the progeny produced by female P. cerealellae on live larvae compared to frozen host larvae that were thawed and held at ambient conditions for up to 96 h, suggesting that live and frozen larvae of C. maculatus are equally suitable for rearing P. cerealellae. However, the data showed that progeny production on frozen hosts gradually declined with thawing duration and was significantly reduced at the thawing duration of 120 h. When live and frozen host larvae were simultaneously presented together to female P. cerealellae at different exposure periods, relatively greater progeny production was recorded on live hosts than on frozen hosts at 12, 24, and 48 h of exposure. This may suggest preference of female P. cerealellae for live versus frozen host larvae. These results are discussed in relation to the life history strategy and host location behavior of P. cerealellae, and may have practical implications in the development of efficient mass rearing systems for the parasitoid.     

High frequency of extrapair copulation with low level of extrapair fertilization in the Lanyu scops owl Otus elegans botelensis

The genetic mating system is known for only a few species of owls. Most of them are genetically monogamous. The Lanyu scops owl Otus elegans botelensis breeds in high density in the forests of Lanyu (Orchid Island), southeast of Taiwan. Because extra‐pair copulations (EPCs) have frequently been observed, we suspected a high degree of extra‐pair fertilizations (EPFs). Through intensive field observations we quantified both within‐pair and extra‐pair copulations of this owl, and used a set of microsatellite loci to assign the parentage of 200 offspring from 108 families collected between 1999 and 2004. Among copulations for which we could identify both participants, 19.77% were EPC. However, no EPC was observed in the period just before egg laying. We found only two cases of parentage mismatch among 108 broods. The first case was most likely a case of brood usurpation, which would be the first case reported in the Strigidae. The second case was an EPF resulting in one offspring. Our study found that parentage mismatch in the Lanyu scops owls to be extremely low, therefore reproductive success can be estimated accurately by simply counting chicks in nests.     

Potential for integrated control of Culex quinquefasciatus (Diptera: Culicidae) using larvicides and guppies

Acute toxicity tests were carried out to determine the effect of three larvicides, spinosad, pirimiphos methyl, and chlorpyrifos, on Culex quinquefasciatus Say, Anopheles gambiae s.s. Giles (Diptera: Culicidae), and guppies, Poecilia reticulata Peters (Pisces: Poeciliidae). Thereafter, larvae of Cx. quinquefasciatus were introduced to P. reticulata in containers of different volumes with low concentrations of each larvicide at established predator to prey densities of 1–35; 5–70 and 10–350 (fish to larvae) respectively. The experiment was replicated six times, and the larval consumption was counted after 24 h. Spinosad and pirimiphos methyl were significantly toxic to Cx. quinquefasciatus, the less susceptible mosquito species. Guppy consumption of Culex larvae was highest at a predator to prey density 5–70. Feeding activity of guppies increased in the spinosad treatment at 49 μg L⁻¹ compared to control and the synthetic larvicides. The synthetic larvicides generally reduced consumption of larvae except for the lowest concentration of pirimiphos methyl which increased it at the highest predator to prey density in a 3 L container. The highest percentage of Culex larvae was consumed by fish in the presence of spinosad at 49 μg L⁻¹ and a predator to prey density of 5–70 in 0.5 L plastic containers. Thus, predator to prey density, container size, type and concentration of larvicide are all important factors to be considered in integrated management of mosquito larvae.     

Influence of inoculation with plant growth promoting rhizobacteria (PGPR) on tomato plant growth and nematode reproduction under greenhouse conditions

Numerous species of soil bacteria which flourish in the rhizosphere of plants or around plant tissues stimulate plant growth and reduce nematode population by antagonistic behavior. These bacteria are collectively known as PGPR (plant growth promoting rhizobacteria). The effects of six isolates of PGPR Pseudomonas putida, Pseudomonas fluorescens, Serratia marcescens, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus cereus, were studied on tomato plant growth and root knot nematode reproduction after 45 days from nematode infection. The highest number of shoot dry weight/g (43.00 g) was detected in the plant treated with S. marcescens; then P. putida (34.33 g), B. amyloliquefaciens (31.66 g), P. fluorescens (30.0 g), B. subtilis (29.0 g), B. cereus (27.0 g) and nematode alone (untreated) 20 g/plant. While the highest number of plant height was observed when plant was treated with S. marcescens, P. fluorescens, P. putida, B. amyloliquefaciens and P. putida 52.66, 50.66, 48 and 48 cm respectively. No significant differences were seen between previous treatments but only had significant differences compared with untreated plant. The highest number of fruit/plant was observed when plants were treated with S. marcescens (10.66), then B. amyloliquefaciens (8.66), P. putida (8), P. fluorescens (8) and B. cereus (7.66). No significant differences between the last 4 treatments, but all had significant differences compared with untreated plants. The highest weight of plant yield (g) was observed with S. marcescens (319.6 g/plant) and the lowest weight of plant yield was observed in plants treated with nematode alone (untreated). On the other hand, the lowest numbers of J₂/10 g of soil (78), galls/root, (24.33) galls/root, egg masses/root (12.66) and egg/egg masses were observed in the plants treated with S. marcescens.     

The effect of Achyranthes japonica extract on larval survival and development and oviposition behavior of Plutella xylostella L. (Lepidoptera: Plutellidae)

The extract of Achyranthes japonica was tested for effects on larval survival and development and the oviposition behavior of the diamondback moth, Plutella xylostella L. Chinese cabbage dipped in A. japonica extract solution showed 51–80% antifeedant activity for 5 days against P. xylostella larvae, and more larvae were also on untreated cabbage leaves 24 h after release. The mortality of P. xylostella larvae increased proportionally to the duration of dipping time in the extract, and both pupation and emergence rates of larvae feeding only on treated cabbage were lower than those for larvae raised on untreated or with a choice of cabbage. The 20-hydroxyecdysone (20E) concentration in leaves was approximately 549, 1232, 1275, and 1426 μg/g at 6, 12, 24, and 48 h after dipping treatment, respectively. Notably, naive females laid more eggs on untreated cabbage than on treated cabbage, and females from larvae raised on treated Chinese cabbage also preferred the non-treated leaves. Our results are in contrast to those from earlier studies using various insect models that confirmed most females prefer to lay eggs on the host type that was eaten in the larval stage (Hopkins host selection principle). Cabbage dipped in the A. japonica solution for 24 h caused 59% larval mortality and inhibited both pupation and emergence rates of the larvae when exposed to plants 15 and 22 days after planting in the field, with the 20E concentration in the treated cabbage leaves at 1600.9 ± 122.36 and 1386.8 ± 24.69 μg/g, respectively. Therefore, the biological effectiveness could be attributed to the 20E in the treated cabbage leaves.     

Horseradish peroxidase production from Spodoptera frugiperda larvae: A simple and inexpensive method

Horseradish peroxidase is used in many biotechnological fields including diagnostics, biocatalysts and biosensors. Horseradish peroxidase isozyme C (HRPC) was extracellularly expressed in Spodoptera frugiperda Sf9 cell culture and in intact larvae. At day 6 post-infection, the concentration of active HRPC in suspension cultures was 3.0 ± 0.1 μg per 1 × 10⁶ cells or 3.0 ± 0.1 mg l⁻¹ with a multiplicity of infection of 1 in the presence of 7.2 μM hemin. Similar yields were obtained in monolayer cultures. In larvae, the HRPC expression level was 137 ± 17 mg HRPC kg⁻¹ larvae at day 6 post-infection with a single larvae thus producing approximately 41 μg HRPC. The whole larval extract was separated by ion exchange chromatography and HRPC was purified in a single step with a yield of 75% and a purification factor of 117. The molecular weight of recombinant HRPC was 44,016 Da, and its glycosylation pattern agreed with that expected for invertebrates. The K_m and V_max were 12.1 ± 1.7 mM and 2673 ± 113 U mg⁻¹, respectively, similar to those of HRP purified from Armoracia rusticana roots. The method described in this study, based on overexpression of HRPC in S. frugiperda larvae, is a simple and inexpensive way to obtain high levels of active enzyme for research and other biotechnological applications.     

Assessment of germination and carnivorous activities of a nematode-trapping fungus Arthrobotrys dactyloides in fungistatic and fungicidal soil environment

Carnivorism is the ability of nematode-trapping fungi to trap and digest the nematodes by sophisticated devices called traps. Delivery of nematode-trapping fungi in soil for bio-control of pest nematodes often fails or gives inconsistent results. Possible reasons for failure could be the effect of soil fungistasis on germination of nematode-trapping fungi in soil environment, use of avirulent species and sensitivity of these fungi to fungicidal residues in soil. Exploitation of nematode-trapping fungi for nematode control demands that it be compatible with fungicides applied in soil or crops and proliferate in soil. This investigation represents is one of the first to evaluate the effect of fungicides on the nematode-trapping fungus Arthrobotrys dactyloides. A. dactyloides showed in vitro carnivorous potential against Meloidogyne incognita, Meloidogyne javanica, Meloidogyne graminicola, Helicotylenchus dihystera and Heterodera cajani. Conidia of A. dactyloides exposed to agricultural soils showed poor germination but formed conidial traps, which captured and killed the soil nematodes. Conidial traps, which trapped the nematodes, grew well in all soils after killing and nutrient absorption from nematode body. Soil amended with 20 mg ai kg⁻¹ of carbendazim and thiram, 30 mg ai kg⁻¹ of mancozeb, 50 mg ai kg⁻¹ of captan, and 100 mg ai kg⁻¹ of carboxin completely checked the conidial trap formation and nematode capturing. 30, 50 and 100 mg ai kg⁻¹ of metalaxyl adversely affected the conidial trap formation and nematode capturing in soil. Propiconazole inhibited 15.2% conidial trap formation up to 50 mg ai kg⁻¹ but caused 93.3% inhibition of conidial traps formation and complete inhibition of nematode capturing at 100 mg ai kg⁻¹. Sulphur, triademefon, and tricyclazole showed least toxic effect on conidial trap formation and nematode capturing activities of A. dactyloides in soil up to 100 mg ai kg⁻¹.