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1.  A Simple Method for Determining Aphelenchoides besseyi Infestation Level of Oryza sativa Seeds 
Journal of Nematology  1999;31(4S):641-643.
A simple extraction method was developed for obtaining the white tip nematode, Aphelenchoides besseyi Christie, from single seeds of rice, Oryza sativa. The method was as follows: Individual rice seeds were split longitudinally and then transferred into single pipet tips. Tips containing a split seed were then singly placed upright in glass vials with water to extract the nematodes. This method was more efficient than the Baermann funnel technique and allowed nearly 100% recovery of living A. besseyi from single rice seeds within 4 hours.
PMCID: PMC2620416  PMID: 19270929
Aphelenchoides besseyi; assay; extraction; methodology; nematode; nematode load; Oryza sativa; rice; white tip disease
2.  Lethal Temperature for Pinewood Nematode, Bursaphelenchus xylophilus, in Infested Wood Using Microwave Energy 
Journal of Nematology  2010;42(2):101-110.
To reduce the risks associated with global transport of wood infested with pinewood nematode Bursaphelenchus xylophilus, microwave irradiation was tested at 14 temperatures in replicated wood samples to determine the temperature that would kill 99.9968% of nematodes in a sample of ≥ 100,000 organisms, meeting a level of efficacy of Probit 9. Treatment of these heavily infested wood samples (mean of > 1,000 nematodes/g of sapwood) produced 100% mortality at 56 °C and above, held for 1 min. Because this “brute force” approach to Probit 9 treats individual nematodes as the observational unit regardless of the number of wood samples it takes to treat this number of organisms, we also used a modeling approach. The best fit was to a Probit function, which estimated lethal temperature at 62.2 (95% confidence interval 59.0-70.0) °C. This discrepancy between the observed and predicted temperature to achieve Probit 9 efficacy may have been the result of an inherently limited sample size when predicting the true mean from the total population. The rate of temperature increase in the small wood samples (rise time) did not affect final nematode mortality at 56 °C. In addition, microwave treatment of industrial size, infested wood blocks killed 100% of > 200,000 nematodes at ≥ 56 °C held for 1 min in replicated wood samples. The 3rd-stage juvenile (J3) of the nematode, that is resistant to cold temperatures and desiccation, was abundant in our wood samples and did not show any resistance to microwave treatment. Regression analysis of internal wood temperatures as a function of surface temperature produced a regression equation that could be used with a relatively high degree of accuracy to predict internal wood temperatures, under the conditions of this study. These results provide strong evidence of the ability of microwave treatment to successfully eradicate B. xylophilus in infested wood at or above 56 °C held for 1 min.
PMCID: PMC3380472  PMID: 22736846
Pinewood nematode; quarantine; microwave; dielectric heating; international trade; embargo; eradication; Probit 9; International Standard of Phytosanitary Measures No. 15
3.  Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species 
Journal of Nematology  2007;39(4):343-355.
A PCR-based diagnostic assay was developed for early detection and identification of Aphelenchoides fragariae directly in host plant tissues using the species-specific primers AFragFl and AFragRl that amplify a 169-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA. These species-specific primers did not amplify DNA from Aphelenchoides besseyi or Aphelenchoides ritzemabosi. The PCR assay was sensitive, detecting a single nematode in a background of plant tissue extract. The assay accurately detected A. fragariae in more than 100 naturally infected, ornamental plant samples collected in North Carolina nurseries, garden centers and landscapes, including 50 plant species not previously reported as hosts of Aphelenchoides spp. The detection sensitivity of the PCR-based assay was higher for infected yet asymptomatic plants when compared to the traditional, water extraction method for Aphelenchoides spp. detection. The utility of using NaOH extraction for rapid preparation of total DNA from plant samples infected with A. fragariae was demonstrated.
PMCID: PMC2586516  PMID: 19259510
Aphelenchoides fragariae; detection; diagnosis; foliar nematode; ITS1; method; NaOH; ornamental host; PCR; rDNA
4.  Seasonal Fluctuations of Soil and Tissue Populations of Ditylenchus dipsaci and Aphelenchoides ritzemabosi in Alfalfa 
Journal of Nematology  1999;31(1):27-36.
Population dynamics of A. ritzemabosi and D. dipsaci were studied in two alfalfa fields in Wyoming. Symptomatic stem-bud tissue and root-zone soil from alfalfa plants exhibiting symptoms of D. dipsaci infection were collected at intervals of 3 to 4 weeks. Both nematodes were extracted from stem tissue with the Baermann funnel method and from soil with the sieving and Baermann funnel method. Soil moisture and soil temperature at 5 cm accounted for 64.8% and 61.0%, respectively, of the variability in numbers of both nematodes in soil at the Big Horn field. Also at the Big Horn field, A. ritzemabosi was found in soil on only three of the 14 collection dates, whereas D. dipsaci was found in soil on 12 dates. Aphelenchoides ritzemabosi was found in stem tissue samples on 9 of the 14 sampling dates whereas D. dipsaci was found on all dates. Populations of both nematodes in stem tissue peaked in October, and soil populations of both peaked in January, when soil moisture was greatest. Numbers of D. dipsaci in stem tissue were related to mean air temperature 3 weeks prior to tissue collection, while none of the climatic factors measured were associated with numbers of A. ritzemabosi. At the Dayton field, soil moisture plus soil temperature at 5 cm accounted for 98.2% and 91.4% of the variability in the soil populations of A. ritzemabosi and D. dipsaci, respectively. Aphelenchoides ritzemabosi was extracted from soil at two of the five collection dates, compared to extraction of D. dipsaci at three dates. Aphelenchoides ritzemabosi was collected from stem tissue at six of the seven sampling dates while D. dipsaci was found at all sampling dates. The only environmental factor that was associated with an increase in the numbers of both nematodes in alfalfa stem tissue was total precipitation 1 week prior to sampling, and this occurred only at the Dayton field. Numbers of A. ritzemabosi in stem tissue appeared to be not affected by any of the environmental factors studied, while numbers of D. dipsaci in stem tissue were associated with cumulative monthly precipitation, snow cover at time of sampling, and the mean weekly temperature 3 weeks prior to sampling. Harvesting alfalfa reduced the numbers of A. ritzemabosi at the Big Horn field and both nematodes at the Dayton field.
PMCID: PMC2620350  PMID: 19270872
alfalfa; alfalfa stem nematode; Aphelenchoides ritzemabosi; chrysanthemum foliar nematode; climate; distribution; Ditylenchus dipsaci; Medicago sativa; nematode; sampling; seasonal fluctuations
5.  Occurrence of Aphelenchoides besseyi in Louisiana Rice Seed and Its Interaction with Sclerotium oryzae in Selected Cultivars 
Journal of Nematology  1984;16(1):65-68.
Aphelenchoides besseyi, the nematode causal agent of white-tip disease of rice, was recovered from 5.5% of 474 seed samples obtained from rice seed warehouses in Louisiana. Laboratory tests in which A. besseyi-infested rice seed was treated with Phostoxin®, a compound used for control of insects in stored grain, indicate that it also has nematicidal properties. In 18-week-duration greenhouse tests, populations of A. besseyi increased 4-5-fold on the cultivars Saturn and Melrose and 3-fold on Nova '76. Green weights of Nova '76 plants inoculated with A. besseyi and Sclerotium oryzae, the causal agent of rice stem rot, were significantly reduced below those of plants inoculated with either organism alone or with distilled water. Weights of Melrose plants were reduced significantly by treatments with A. besseyi alone and A. besseyi plus S. oryzae, but not by S. oryzae alone. Saturn plant weights were not reduced significantly by either organism alone or by the two in combination.
PMCID: PMC2618349  PMID: 19295876
Oryza sativa; host suitability; nematode-fungus interaction; white-tip disease; chemical control
6.  Nematode Community Structure in Desert Soils: Nematode Recovery 
Journal of Nematology  1975;7(4):343-346.
The sugar-flotation-sieving (SFS) and Baermann-funnel (BF) methods were compared for nematode extraction efficiency. The SFS method recovered nematodes from more trophic groups whereas greater total numbers of individuals were recovered by BF. In a test to validate the efficiency of SFS, virtually 100% of the nematodes added to desert soil prior to extraction were recovered by four consecutive SFS washings of each soil sample. Estimations of nematode biomass in desert soils based on numbers of nematodes extracted by the two methods were similar unless there was large reserve of eggs in the soil. The biomass of nematodes from a Colorado desert soil was 0.9 g/m² as determined by both methods, whereas BF gave 0.17 g/m² for nematodes from a Mojave desert soil as compared to 0.9 g/m² with SFS.
PMCID: PMC2620136  PMID: 19308179
soil sampling
7.  Plant-parasitic Nematode Problems in the Pacific Islands 
Journal of Nematology  1988;20(2):173-183.
The Pacific islands have a diverse range of food and cash crops with indigenous and introduced nematode problems. The staple food crops have serious nematode pests, such as Meloidogyne spp. on sweet potato, Hirschmanniella miticausa causing corm rot of taro, and Pratylenchus coffeae and Radopholus sp. producing tuber dry rot of yams. Bananas are infested with P. coffeae or R. similis, citrus with Tylenchulus semipenetrans, rice with Aphelenchoides besseyi, and ginger with Meloidogyne spp. and R. similis. Rotylenchulus reniformis, P. zeae, P. brachyurus, and Helicotylenchus spp. are important on all of these and other crops, such as sugarcane, passion fruit, pawpaw, and cassava. Meloidogyne spp. cause serious damage to local and introduced leaf and fruit vegetables and other crops, such as tobacco, sugarcane, pawpaw, black pepper, and pyrethrum. Many other plant-parasitic genera and species, some undescribed, occur in the Pacific, and there are many islands still to be investigated.
PMCID: PMC2618799  PMID: 19290200
Pacific islands; plant-parasitic nematode
8.  Occurrence of the Clover Cyst Nematode, Heterodera trifolii, in Prince Edward Island Soils 
Journal of Nematology  1993;25(4S):876-879.
In a survey of potato and rotational crops on Prince Edward Island, Canada, the cyst stage of the clover cyst nematode, Heterodera trifolii, was found in 43 of 63 sites sampled; however, only 12% of the cysts contained eggs. The root lesion nematode, Pratylenchus penetrans, was the dominant plant parasitic nematode and was found in 56 sites. Extraction of cysts from soil was similar using either the Schuiling centrifuge or the Fenwick can method, although the former was more convenient to use. The modified Baermann funnel method was not efficient for detecting the clover cyst nematode in soil.
PMCID: PMC2619464  PMID: 19279856
Baermann funnel; clover cyst nematode; genwick can; Heterodera trifolii; method; nematode; Pratylenchus penetrans; root lesion nematode; Schuiling centrifuge; survey
9.  A Comparative Analysis of Extraction Methods for the Recovery of Anguina sp. from Grass Seed Samples 
Journal of Nematology  1999;31(4S):635-640.
Four procedures were compared in their efficacy to extract juveniles of Anguina agrostis from commercial grass seed. The procedures included those currently used by the state regulatory laboratories of Oregon and California, as well as new tests developed to determine juvenile viability for the phytosanitary certification of fumigated grass seed. Eleven seed lots of Agrostis tenuis (bentgrass) and Dactylis glomerata (orchardgrass) naturally infested with varying levels of juveniles of Anguina were individually analyzed. Only one procedure, a new live recovery test, yielded nematodes in all 11 samples and is recommended as the best method for use by regulatory agencies. In comparison, although the other three extraction procedures resulted in greater numbers of Anguina agrostis juveniles per gram of seed, they failed to yield any nematodes in as many as four seed lots with low infection levels.
PMCID: PMC2620417  PMID: 19270928
Anguina agrostis; assay; bentgrass; extraction procedures; nematode; orchardgrass; phytosanitary certification; regulatory nematology; seed-borne nematodes; technique
10.  Guidelines for Introducing Beneficial Insect-parasitic Nematodes into the United States 
Journal of Nematology  1988;20(Annals 2):50-56.
Guidelines are suggested to implement the introduction of beneficial insect-parasitic nematodes into the United States from abroad. These suggestions result from experiences and research with these and other biological control agents and from the current need for procedures to import nematodes. Subjects considered are need to import, foreign exploration, taxonomy, shipment, quarantine facilities, permits, host range tests, release, and documentation. Nematodes covered under these suggested guidelines include entomopathogenic species of mermithids, sphaerulariids, aphelenchids, steinernematids, and heterorhabditids. Host specificity and safety tests are discussed. Concern over the possible concomitant introduction of plant-parasitic nematodes, insects, or other pests is expressed. Current information on the treatment of insect-parasitic nematodes by the Environmental Protection Agency and USDA's Animal and Plant Health Inspection Service is presented. These suggested guidelines are presented to stimulate the development of workable protocols for safe introduction of beneficial insect-parasitic nematodes into the United States from aboard.
PMCID: PMC2618865  PMID: 19290303
biological control; entomogenous nematode; guideline; insect parasite; introduction; nematode introduction; permit form; procedure
11.  Comparison of Extraction and Shipping Methods for Cysts and Juveniles of Heterodera glycines 
Journal of Nematology  1997;29(1):127-132.
Experiments to determine the effects of extraction techniques and the influence of shipping on extraction of Heterodera glycines life stages gave variable results. Shipping did not significantly affect numbers of nematodes extracted. More second-stage juveniles (J2) were extracted with Baermann funnels than with an elutriator, probably because incubation of encysted eggs on the Baermann funnel for 1 week allowed hatching to occur. Sieving was more efficient than elutriation for extracting cysts. Adding air agitation to the water pressure during elutriation increased extraction efficiency of cysts but not J2. Sample sizes of 250 cm³ and 500 cm³ did not influence extraction efficiency of cysts; however, sample size did influence extraction of J2.
PMCID: PMC2619757  PMID: 19274141
Baennann funnel; centrifugal flotation; cyst; egg; elutriation; extraction; Heterodera glycines; methods; nematode; second-stage juvenile; shipping; soybean cyst nematode
12.  Post-Control Surveillance of Triatoma infestans and Triatoma sordida with Chemically-Baited Sticky Traps 
Background
Chagas disease prevention critically depends on keeping houses free of triatomine vectors. Insecticide spraying is very effective, but re-infestation of treated dwellings is commonplace. Early detection-elimination of re-infestation foci is key to long-term control; however, all available vector-detection methods have low sensitivity. Chemically-baited traps are widely used in vector and pest control-surveillance systems; here, we test this approach for Triatoma spp. detection under field conditions in the Gran Chaco.
Methodology/Principal Findings
Using a repeated-sampling approach and logistic models that explicitly take detection failures into account, we simultaneously estimate vector occurrence and detection probabilities. We then model detection probabilities (conditioned on vector occurrence) as a function of trapping system to measure the effect of chemical baits. We find a positive effect of baits after three (odds ratio [OR] 5.10; 95% confidence interval [CI95] 2.59–10.04) and six months (OR 2.20, CI95 1.04–4.65). Detection probabilities are estimated at p≈0.40–0.50 for baited and at just p≈0.15 for control traps. Bait effect is very strong on T. infestans (three-month assessment: OR 12.30, CI95 4.44–34.10; p≈0.64), whereas T. sordida is captured with similar frequency in baited and unbaited traps.
Conclusions/Significance
Chemically-baited traps hold promise for T. infestans surveillance; the sensitivity of the system at detecting small re-infestation foci rises from 12.5% to 63.6% when traps are baited with semiochemicals. Accounting for imperfect detection, infestation is estimated at 26% (CI95 16–40) after three and 20% (CI95 11–34) after six months. In the same assessments, traps detected infestation in 14% and 8.5% of dwellings, whereas timed manual searches (the standard approach) did so in just 1.4% of dwellings only in the first survey. Since infestation rates are the main indicator used for decision-making in control programs, the approach we present may help improve T. infestans surveillance and control program management.
Author Summary
Triatoma infestans is the main vector of Chagas disease in southern South America. Dwelling-infesting populations are controlled through insecticide-spraying campaigns; however, dwellings are often re-infested when insecticide effects wane, and this leads to the re-establishment of disease transmission. Detecting and eliminating re-infestation foci is therefore crucial to prevent new cases. Unfortunately, available vector detection methods all have low sensitivity. Here, we show that simple sticky traps baited with widely available chemicals are significantly more sensitive than either unbaited traps or active manual searches by trained staff — the standard method used in control programs. Increased trap sensitivity (about 500% higher), together with an analytical approach that takes detection failures into account, allows us to estimate dwelling infestation rates at about 20–26%; in contrast, just 0–1.4% of dwellings were identified as infested by manual searches. This large difference highlights the importance of enhancing surveillance systems, and reveals how crude infestation indices may mislead decision-makers. We conclude that chemically baited sticky traps can help improve T. infestans surveillance systems and thus strengthen vector control program management.
doi:10.1371/journal.pntd.0001822
PMCID: PMC3441417  PMID: 23029583
13.  Infestation by pyrethroids resistant bed bugs in the suburb of Paris, France 
Parasite  2012;19(4):381-387.
Bed bugs are hematophagous insects responsible for a re-emerging and challenging indoor pest in many countries. Bed bugs infestations may have health consequences including nuisance biting, cutaneous and systemic reactions. This resurgence can probably be attributed to factors such as increased international travel and development of resistance against insecticides. Resistance against pyrethroids has been reported several times from the USA and rarely in Europe. In France, very few data on bed bugs are available. The present study aimed to assess the infestation by bed bugs of a complex of two high-rise apartment buildings in the suburb of Paris and to evaluate their susceptibility to pyrethroid insecticides. We inspected for bed bugs 192 out of 198 apartments units (97%) and interviewed their residents. 76 (39.6%) apartments were infested. Among the 97 residents living in infested apartments, 53 (54.6%) reported bed bug bites. A total of 564 bed bugs were collected in the infested units. Bioassays showed that 54 out of 143 bed bugs were resistant to pyrethroids (37.8%; 95% confidence interval: 29.9-45.7%). DNA sequencing showed that all bed bugs tested (n = 124) had homozygous L925I kdr-like gene mutation. The level of pyrethroid resistance found indicates that this phenomenon was already established in the site and prompts the need to reevaluate the wide use of pyrethroids to control bed bugs.
doi:10.1051/parasite/2012194381
PMCID: PMC3671460  PMID: 23193523
bed bugs; Cimex lectularius; pyrethroids; insecticide; resistance; punaises de lit; Cimex lectularius; pyréthrinoïdes; insecticide; résistance
14.  Transcriptomic Analysis of the Rice White Tip Nematode, Aphelenchoides besseyi (Nematoda: Aphelenchoididae) 
PLoS ONE  2014;9(3):e91591.
Background
The rice white tip nematode Aphelenchoides besseyi, a devastating nematode whose genome has not been sequenced, is distributed widely throughout almost all the rice-growing regions of the world. The aims of the present study were to define the transcriptome of A. besseyi and to identify parasite-related, mortality-related or host resistance-overcoming genes in this nematode.
Methodology and Principal Findings
Using Solexa/Illumina sequencing, we profiled the transcriptome of mixed-stage populations of A. besseyi. A total of 51,270 transcripts without gaps were produced based on high-quality clean reads. Of all the A. besseyi transcripts, 9,132 KEGG Orthology assignments were annotated. Carbohydrate-active enzymes of glycoside hydrolases (GHs), glycosyltransferases (GTs), carbohydrate esterases (CEs) and carbohydrate-binding modules (CBMs) were identified. The presence of the A. besseyi GH45 cellulase gene was verified by in situ hybridization. Given that 13 unique A. besseyi potential effector genes were identified from 41 candidate effector homologs, further studies of these homologs are merited. Finally, comparative analyses were conducted between A. besseyi contigs and Caenorhabditis elegans genes to look for orthologs of RNAi phenotypes, neuropeptides and peptidases.
Conclusions and Significance
The present results provide comprehensive insight into the genetic makeup of A. besseyi. Many of this species' genes are parasite related, nematode mortality-related or necessary to overcome host resistance. The generated transcriptome dataset of A. besseyi reported here lays the foundation for further studies of the molecular mechanisms related to parasitism and facilitates the development of new control strategies for this species.
doi:10.1371/journal.pone.0091591
PMCID: PMC3956754  PMID: 24637831
15.  Effects of Storage Temperature and Extraction Procedure on Recovery of Plant-parasitic Nematodes from Field Soils 
Journal of Nematology  1969;1(3):240-247.
Storage of nematodes in soil at -15 C for 1 to 16 weeks greatly increased nematode recovery by a sugar-flotation-sieving procedure. One week of exposure to -15 C killed all nematodes except Pratylenchus zeae and Tylenchorhynchus claytoni which were recoverable in decreasing numbers up to 10 weeks by the Baermann funnel method. Optimum storage temperature for survival of most nematode species was 13 C. The numbers of Meloidogyne incognita, T. claytoni, Belonolaimus Iongicaudatus, and P. zeae recoverable by either extraction method remained constant or increased when stored at 13-24 C for 16 weeks. This was also true for Helicotylenchtts dihystera and Xiphinema americanum extracted by the Baermann funnel technique, whereas the numbers retrieved by the sugar-flotation-sieving method decreased slightly. All species except T. claytoni decreased appreciably in soil stored at 36 C.
PMCID: PMC2617830  PMID: 19325684
16.  Sensitive PCR Detection of Meloidogyne arenaria, M. incognita, and M. javanica Extracted from Soil 
Journal of nematology  2006;38(4):434-441.
We have developed a simple PCR assay protocol for detection of the root-knot nematode (RKN) species Meloidogyne arenaria, M. incognita, and M. javanica extracted from soil. Nematodes are extracted from soil using Baermann funnels and centrifugal flotation. The nematode-containing fraction is then digested with proteinase K, and a PCR assay is carried out with primers specific for this group of RKN and with universal primers spanning the ITS of rRNA genes. The presence of RKN J2 can be detected among large numbers of other plant-parasitic and free-living nematodes. The procedure was tested with several soil types and crops from different locations and was found to be sensitive and accurate. Analysis of unknowns and spiked soil samples indicated that detection sensitivity was the same as or higher than by microscopic examination.
PMCID: PMC2586468  PMID: 19259460
Detection; diagnosis; Meloidogyne arenaria; Meloidogyne incognita; Meloidogyne javanica; PCR; root-knot nematode; soil
17.  Distribution of the Long-Horned Beetle, Dectes texanus, in Soybeans of Missouri, Western Tennessee, Mississippi, and Arkansas 
The long-horned beetle, Dectes texanus LeConte (Coleoptera: Cerambycidae), is a stem-boring pest of soybeans, Glycine max (L.) Merrill (Fabales: Fabaceae). Soybean stems and stubble were collected from 131 counties in Arkansas, Mississippi, Missouri, and Tennessee and dissected to determine D. texanus infestation rates. All states sampled had D. texanus present in soybeans. Data from Tennessee and Arkansas showed sample infestations of D. texanus averaging nearly 40%. Samples from Missouri revealed higher infestation in the twelve southeastern counties compared to the rest of the state. Data from Mississippi suggested that D. texanus is not as problematic there as in Arkansas, Missouri, and Tennessee. Infestation rates from individual fields varied greatly (0–100%) within states. In Tennessee, second crop soybeans (i.e. soybeans planted following winter wheat) had lower infestations than full season soybeans. A map of pest distribution is presented that documents the extent of the problem, provides a baseline from which changes can be measured, contributes data for emergency registration of pesticides for specific geographic regions, and provides useful information for extension personnel, crop scouts, and growers.
doi:10.1673/031.010.14138
PMCID: PMC3016958  PMID: 21062147
Glycine max; distribution
18.  A Rapid Decision Sampling Plan for Implementing Area—Wide Management of the Red Palm Weevil, Rhynchophorus ferrugineus, in Coconut Plantations of India 
The red palm weevil Rhynchophorus ferrugineus Olivier (Curculionidae/Rhynchophoridae/Dryophthoridae) is a lethal pest of young coconut palms, Cocos nucifera L. (Arecales: Arecaceae), with a highly aggregated population distribution pattern. R. ferrugineus is managed in several coconut growing countries using area-wide pheromone based programmes that need a substantial commitment of funds over a period of time. Often, decisions to implement area-wide management of R. ferrugineus are based on pheromone trap captures in surveillance traps and or infestation reports. Implementing area-wide management of this pest on the basis of such data can be inaccurate, as it may either under or over estimate the pest intensity in the field. This study presents sampling plans for rapid and accurate classification of R. ferrugineus infestation in coconut plantations of India by inspecting palms to detect infestation in a sequence until a decision to either implement or not to initiate area-wide management of R. ferrugineus can be made. The sampling plans are based on a common aggregation index of 3.45, assumed action threshold values of either 1.0 (plan A) or 0.5 (plan B) per cent infested palms and a risk factor of making the wrong decision set at 0.05. Using plans A and B, if the cummulative number of infested palms in a young 1 hectare coconut plantation is zero out of 150 palms for both plans, then area-wide management is not required, while on the other hand, if the cummulative number of infested palms for the same area is 6 (plan A), or 5 (plan B), then area-wide management of R. ferrugineus is essential. The proposed sampling plans are efficient tools in decision making, particularly at very low and high levels of infestation and can also be used to assess the performance of R. ferrugineus IPM programmes that are in progress. These plans not only save time and money as only a small area needs to be sampled to arrive at a correct decision, but are also efficient in rating the infestation level accurately.
doi:10.1673/031.008.1501
PMCID: PMC3061587  PMID: 20337561
sequential sampling; Cocos nucifera
19.  Two Simple Methods for the Collection of Individual Life Stages of Reniform Nematode, Rotylenchulus reniformis 
Journal of Nematology  2013;45(2):87-91.
The sedentary semi-endoparasitic nematode Rotylenchulus reniformis, the reniform nematode, is a serious pest of cotton and soybean in the United States. In recent years, interest in the molecular biology of the interaction between R. reniformis and its plant hosts has increased; however, the unusual life cycle of R. reniformis presents a unique set of challenges to researchers who wish to study the developmental expression of a particular nematode gene or evaluate life stage–specific effects of a specific treatment such as RNA-interference or a potential nematicide. In this report, we describe a simple method to collect R. reniformis juvenile and vermiform adult life stages under in vitro conditions and a second method to collect viable parasitic sedentary females from host plant roots. Rotylenchulus reniformis eggs were hatched over a Baermann funnel and the resultant second-stage juveniles incubated in petri plates containing sterile water at 30°C. Nematode development was monitored through the appearance of fourth-stage juveniles and specific time-points at which each developmental stage predominated were determined. Viable parasitic sedentary females were collected from infected roots using a second method that combined blending, sieving, and sucrose flotation. Rotylenchulus reniformis life stages collected with these methods can be used for nucleic acid or protein extraction or other experimental purposes that rely on life stage–specific data.
PMCID: PMC3700741  PMID: 23833322
host-parasitic relationship; life stages; reniform nematode; Rotylenchulus reniformis; technique
20.  Modeling Disease Vector Occurrence when Detection Is Imperfect: Infestation of Amazonian Palm Trees by Triatomine Bugs at Three Spatial Scales 
Background
Failure to detect a disease agent or vector where it actually occurs constitutes a serious drawback in epidemiology. In the pervasive situation where no sampling technique is perfect, the explicit analytical treatment of detection failure becomes a key step in the estimation of epidemiological parameters. We illustrate this approach with a study of Attalea palm tree infestation by Rhodnius spp. (Triatominae), the most important vectors of Chagas disease (CD) in northern South America.
Methodology/Principal Findings
The probability of detecting triatomines in infested palms is estimated by repeatedly sampling each palm. This knowledge is used to derive an unbiased estimate of the biologically relevant probability of palm infestation. We combine maximum-likelihood analysis and information-theoretic model selection to test the relationships between environmental covariates and infestation of 298 Amazonian palm trees over three spatial scales: region within Amazonia, landscape, and individual palm. Palm infestation estimates are high (40–60%) across regions, and well above the observed infestation rate (24%). Detection probability is higher (∼0.55 on average) in the richest-soil region than elsewhere (∼0.08). Infestation estimates are similar in forest and rural areas, but lower in urban landscapes. Finally, individual palm covariates (accumulated organic matter and stem height) explain most of infestation rate variation.
Conclusions/Significance
Individual palm attributes appear as key drivers of infestation, suggesting that CD surveillance must incorporate local-scale knowledge and that peridomestic palm tree management might help lower transmission risk. Vector populations are probably denser in rich-soil sub-regions, where CD prevalence tends to be higher; this suggests a target for research on broad-scale risk mapping. Landscape-scale effects indicate that palm triatomine populations can endure deforestation in rural areas, but become rarer in heavily disturbed urban settings. Our methodological approach has wide application in infectious disease research; by improving eco-epidemiological parameter estimation, it can also significantly strengthen vector surveillance-control strategies.
Author Summary
Blood-sucking bugs of the genus Rhodnius are major vectors of Chagas disease. Control and surveillance of Chagas disease transmission critically depend on ascertaining whether households and nearby ecotopes (such as palm trees) are infested by these vectors. However, no bug detection technique works perfectly. Because more sensitive methods are more costly, vector searches face a trade-off between technical prowess and sample size. We compromise by using relatively inexpensive sampling techniques that can be applied multiple times to a large number of palms. With these replicated results, we estimate the probability of failing to detect bugs in a palm that is actually infested. We incorporate this information into our analyses to derive an unbiased estimate of palm infestation, and find it to be about 50% – twice the observed proportion of infested palms. We are then able to model the effects of regional, landscape, and local environmental variables on palm infestation. Individual palm attributes contribute overwhelmingly more than landscape or regional covariates to explaining infestation, suggesting that palm tree management can help mitigate risk locally. Our results illustrate how explicitly accounting for vector, pathogen, or host detection failures can substantially improve epidemiological parameter estimation when perfect detection techniques are unavailable.
doi:10.1371/journal.pntd.0000620
PMCID: PMC2830460  PMID: 20209149
21.  Human ocular filariasis: further evidence on the zoonotic role of Onchocerca lupi 
Parasites & Vectors  2012;5:84.
Background
Among ocular vector-borne pathogens, Onchocerca volvulus, the agent of the so-called “river blindness”, affects about 37 million people globally. Other Onchocerca spp. have been sporadically reported as zoonotic agents. Cases of canine onchocerciasis caused by Onchocerca lupi are on the rise in the United States and Europe. Its zoonotic role has been suspected but only recently ascertained in a single case from Turkey. The present study provides further evidence on the occurrence of O. lupi infesting human eyes in two patients from Turkey (case 1) and Tunisia (case 2). The importance of obtaining a correct sample collection and preparation of nematodes infesting human eyes is highlighted.
Methods
In both cases the parasites were identified with morpho-anatomical characters at the gross examination, histological analysis and anatomical description and also molecularly in case 1.
Results
The nematode from the first case was obviously O. lupi based on their morphology at the gross examination, histological analysis and anatomical description. In the second case, although the diagnostic cuticular characters were not completely developed, other features were congruent with the identification of O. lupi. Furthermore, the morphological identification was also molecularly confirmed in the Turkish case.
Conclusions
The results of this study suggest that O. lupi infestation is not an occasional finding but it should be considered in the differential diagnosis of other zoonotic helminths causing eye infestation in humans (e.g., D. immitis and Dirofilaria repens). Both cases came from areas where no cases of canine onchocerciasis were previously reported in the literature, suggesting that an in depth appraisal of the infestation in canine populations is necessary. Physicians and ophthalmologists are advised on how to preserve nematode samples recovered surgically, to allow a definitive, correct etiological diagnosis.
doi:10.1186/1756-3305-5-84
PMCID: PMC3407723  PMID: 22541132
Onchocerca lupi; Zoonosis; Ocular infestation; Dog; Turkey, Tunisia
22.  Effect of Controlled Cold Storage on Recovery of Rotylenchulus reniformis from Naturally Infested Soil 
Journal of Nematology  2005;37(3):272-275.
Rotylenchulus reniformis is rapidly becoming the most economically important pest associated with cotton in the southeastern United States. Incentive programs have been implemented to support sampling of production fields to determine the presence and abundance of R. reniformis. These sampling programs have dramatically increased the number of soils samples submitted to nematology laboratories during autumn. The large numbers of samples overwhelm most labs and require placement in cold storage until extraction. Therefore, the objective of this study was to examine the length of time soils infested with R. reniformis can be stored before nematode extraction without compromising the accuracy of estimates of population densities. A sandy loam and a silty loam were the two cotton production soils used in this study. Rotylenchulus reniformis numbers decreased 61%during the first 180 days of storage in both soils. Rotylenchulus reniformis numbers from the initial sampling through 180 days decreased as a linear function. The decline of R. reniformis numbers during storage was estimated as 0.28% of the population lost daily from the maximum population through 180 days. The diminution of nematode numbers from 180 through 1,080 days in storage continued, but at a slower rate. Numbers of R. reniformis declined to less than 89%, 93%, and 99% of the initial population within 360, 720, and 1,080 days, respectively, of storage. The reduction of R. reniformis numbers over 180 days can be adjusted, allowing a more accurate estimation of R. reniformis levels in soil samples stored at 4 °C.
PMCID: PMC2620982  PMID: 19262872
Rotylenchulus reniformis; soil storage; population density
23.  Nematodes in Michigan. I. Distribution of Heterodera glycines and Other Plant-parasitic Nematodes in Soybean 
Journal of Nematology  1994;26(4S):720-726.
In 1992, a detection survey for Heterodera glycines (soybean cyst nematode) was conducted in 16 counties in Michigan. The nematode was detected in 12 counties, with absolute frequencies ranging from 6 to 100%. A total of 149 samples was collected, and 53% were infested with H. glycines. Eighty-four growers participated in the survey, and 38 had samples collected from more than one field. Of the 38 growers, 42% had all samples positive for H. glycines, 18% had some positive and negative fields, and 39% had all negative. A risk index was developed to quantify three types of risks: short-term, long-term, and border risk from neighboring counties. Soybean yield was regressed on H. glycines population density and number of years of soybean. Thirty-one percent of the variability in soybean yields was explained by H. glycines cyst population densities. Total number of years in soybean over the last 20 year period explained 19% of the variability in yields. In addition, H. glycines frequencies and population densities were inversely related to Pratylenchus spp. frequencies and population densities.
PMCID: PMC2619557  PMID: 19279953
community ecology; distribution; Heterodera glycines; Glycine max; Michigan; nematode; Pratylenchus spp.; risk assessment; soybean; soybean cyst nematode
24.  Spatial Patterns of Belonolaimus spp. Among and Within Citrus Orchards on Florida's Central Ridge 
Journal of Nematology  1996;28(3):352-359.
A survey was initiated to determine the incidence of Belonolaimus spp. (sting nematodes) in citrus orchards in the central ridge region of Florida, following widespread damage by these nematodes to young trees replanted after freezing weather in 1989-90. Sting nematodes were detected in 50% of 210 samples and in 64% of 84 orchards surveyed. More orchards in Polk County were infested with sting nematodes (82%) than in counties to the north (36%) or south (48%). Principal component analysis of morphometric data separated six of seven sting nematode populations in northeastern Polk County from six populations in adjacent regions. Stylet:tail ratio for nematodes in northeastern Polk County tend to be > 1.0 and were ≪ 1.0 for all other populations. Patchiness of nematodes within an orchard was associated with stunted trees (23% smaller), reduced root mass density (25% lower), and low fruit yield (57% reduction). Soil texture did not vary among trees of different size in the orchard, but soil water potential between irrigation events was highest beneath small trees with low root mass density. Results of the survey indicate that the incidence of sting nematodes in orchards on the central ridge is much higher than previously estimated and that sting nematodes can cause substantial damage in replanted orchards. Further research is needed to evaluate the significance of sting nematode population variability and its relationship to citrus crop loss in Florida.
PMCID: PMC2619698  PMID: 19277153
Belonolaimus longicaudatus; citrus; crop loss assessment; ecology; nematode; nematode survey; soil moisture; spatial distribution; spatial pattern; sting nematode
25.  Field Efficacy of Furfural as a Nematicide on Turf 
Journal of Nematology  2014;46(1):8-11.
A commercial formulation of furfural was recently launched in the United States as a turfgrass nematicide. Three field trials evaluated efficacy of this commercial formulation on dwarf bermudagrass putting greens infested primarily with Belonolaimus longicaudatus, Meloidogyne graminis, or both these nematodes, and in some cases with Mesocriconema ornatum or Helicotylenchus pseudorobustus. In all these trials, furfural improved turf health but did not reduce population densities of B. longicaudatus, M. graminis, or the other plant-parasitic nematodes present. In two additional field trials, efficacy of furfural at increasing depths in the soil profile (0 to 5 cm, 5 to 10 cm, and 10 to 15 cm) against B. longicaudatus on bermudagrass was evaluated. Reduction in population density of B. longicaudatus was observed in furfural-treated plots for depths below 5 cm on several dates during both trials. However, no differences in population densities of B. longicaudatus were observed between the furfural-treated plots and the untreated control for soil depth of 0 to 5 cm during either trial. These results indicate that furfural applications can improve health of nematode-infested turf and can reduce population density of plant-parasitic nematodes in turf systems. Although the degree to which turf improvement is directly caused by nematicidal effects is still unclear, furfural does appear to be a useful nematode management tool for turf.
PMCID: PMC3957574  PMID: 24644368
Belonolaimus longicaudatus; bermudagrass; Cynodon dactylon; furfural; Helicotylenchus pseudorobustus; management; Meloidogyne graminis; Mesocriconema ornatum; ring nematode; root-knot nematode; spiral nematode; sting nematode; turfgrass

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