The impact of nematode parasites on the behaviour of an Australian lizard,the gidgee skink <Emphasis Type="Italic">Egernia stokesii</Emphasis>

The Australian scincid lizard Egernia stokesii lives in social groups and is infected with two nematode species: Pharyngodon tiliquae and Thelandros trachysauri. This study asked whether those nematodes affected levels of lizard activity in field populations. In a laboratory colony, application of a combination of ivermectin and fenbendazole reduced nematode egg count in lizard scats after 12 weeks. In the field, the same doses of those antihelminthic drugs were applied to lizards in six social groups across three populations, and a saline control was given to lizards in six adjacent groups. Observations showed significant changes in behaviour between the two groups developing over 2 months. Drug-treated lizards spent more time basking and moved about for longer times during observation sessions. The results suggest that nematode infection altered host behaviour and reduced fitness. No influence of social group size was detected on the impact of parasitic nematodes.     

Penetration and Postinfection Development of Meloidogyne incognita on Cotton as Affected by Glomus intraradices and Phosphorus

The influence of the vesicular-arbuscular mycorrhizal fungus Glomus intraradices (Gi) and superphosphate (P) on penetration, development, and reproduction of Meloidogyne incognita (Mi) was studied on the Mi-susceptible cotton cultivar Stoneville 213 in an environmental chamber at 28 C. Plants were inoculated with Mi eggs at planting or after 28 days and destructively sampled 7, 14, 21, and 28 days after nematode inoculation. Mi penetration after 7 days was similar in all treatments at either inoculation interval. At 28 days, however, nematode numbers were least in mycorrhizal root systems and greatest in root systems grown with supplemental P. The rate of development of second-stage juveniles to ovipositing females was unaffected by Gi or P when Mi was added at planting, but was delayed in mycorrhizal root systems when Mi was added 28 days after planting. Nematode reproduction was lower in mycorrhizal than in nonmycorrhizal root systems at both Mi inoculation intervals. Nematode reproduction was stimulated by P when Mi was added at planting, but was similar to reproduction in the low P nonmycorrhizal treatment when Mi was added 28 days after planting. Eggs per female were increased by P fertility when Mi was added at planting.     

Frequency of deposition and location of Baylisascaris procyonis eggs in raccoon feces

Baylisascaris procyonis is a large ascarid nematode found in the small intestine of raccoons (Procyon lotor). Infection with larvae of B. procyonis can produce visceral, ocular, and neural larval migrans in humans. Infected raccoons can shed millions of eggs a day in their feces. However, it is unknown whether eggs are consistently shed or whether eggs occur at irregular intervals by the population of female nematodes within a host. We trapped, infected, and collected daily fecal samples from 11 raccoons maintained in captivity. Eggs from B. procyonis were obtained from anterior, central, and posterior sections of raccoon feces, isolated by flotation, and quantified under 100× magnification. Naturally infected raccoons were collected and used as a comparison with the experimentally infected group. All raccoons in the experimental group (n=11) became infected with B. procyonis after consuming one infected mouse. Additionally, differential egg deposition rates were observed among individual raccoons from the experimental and naturally infected groups. Mean number of eggs per gram of feces (means±SE) was 16,563±4,321, which was less than previously reported for the species. However, no differences (F(2,30)=0.84, P=0.45) were noted in mean number of eggs per gram of feces among fecal sections. Wildlife biologists, veterinarians, health officials, and researchers of B. procyonis should collect daily fecal samples for a minimum of 3 days before identifying a raccoon as negative for B. procyonis infection. However, it does not matter where within the fecal matter the sample is obtained.     

Some properties of a pheromone allowing individual recognition, from the scats of an Australian lizard, Egernia striolata

  The Australian lizard, Egernia striolata, can distinguish its own scats from those of unfamiliar conspecific individuals. This appears to be unrelated to diet, because there is no difference in the response to scats from unfamiliar lizards fed on diets that are the same or different from the test lizard. The signal that induces the response is not a visual or tactile property of scat structure, because test lizards respond equally to crushed and intact scats. We suggest that a pheromone is secreted onto the scat as it is produced. Water solutions of scats did not contain signal components that allowed lizards to distinguish their own scats from others. However, solutions of scats in dichloromethane (DCM) retained unique characteristics, and test lizards responded more strongly to the solution from scats of an unfamiliar lizard that to the solution from their own scats. Further fractionation of the DCM solution in pentane and in methanol led to loss of the unique signals needed for individual recognition, but those were restored when the pentane and methanol fractions were recombined. We infer that these lizards can distinguish between scats of different individuals on the basis of signals they receive from a complex combination of chemicals. Received: 14 December 1998 / Received in revised form: 9 April 1999 / Accepted: 12 April 1999     

Three new species of Huffmanela Moravec, 1987 (Nematoda: Trichosomoididae) from the gills of marine fish off New Caledonia

Three new species of Huffmanela, here described from eggs only, are reported from the gills of marine fish caught off Nouméa, New Caledonia. Eggs of Huffmanela branchialis n. sp., from Nemipterus furcosus (Nemipteridae), are 45-52 (mean 48) microm in length and 23-30 (mean 25) microm in width, with thin shells. Each egg is enclosed in a thin membrane forming a spindle-shaped envelope 53-85 (mean 63) microm in length. Eggs of H. filamentosa n. sp., from Gymnocranius grandoculis (Lethrinidae), are 48-53 (mean 50) microm in length and 25-30 (mean 27) microm in width, with thin shells. Each egg bears a few long (150 microm), thin filaments. Eggs of these two new species were compared to those of H. paronai Moravec & Garibaldi, 2000, which are redescribed. Eggs of H. ossicola n. sp. were found within the branchial arch bone of Bodianus loxozonus (Labridae) and also filled the spinal chord bone and other bones. This is the first species of Huffmanela reported from bone tissue. Eggs are large, 72-88 (mean 79) microm in length and 32-40 (mean 36) microm in width, with a very thick shell. Each egg is covered with numerous filaments enclosed in a thin envelope. Fresh eggs were unembryonated, but embryos were visible after incubation in seawater. The three new species can be distinguished from other species of Huffmanela by size and the nature of the egg covering. Egg morphology of and their location in the host suggest different life-cycles: those of the first two species (small eggs, thin shells, egg covering possibly favouring flotation) are released from the gill mucosa with the turnover of living tissues and immediately continue their life-cycle, but eggs of H. ossicola (large eggs, thick shell) are only available for the continuation of the life-cycle after the host's death.     

Dispersal of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in an urban endemic dengue area in the State of Rio de Janeiro,Brazil

Experimental releases of female Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus were performed in August and September 1999, in an urban area of Nova Igua u, State of Rio de Janeiro, Brazil, to estimate their flight range in a circular area of 1,600 m where 1,472 ovitraps were set. Releases of 3,055 Ae. aegypti and 2,225 Ae. albopictus females, fed with rubidium (Rb)-marked blood and surgically prevented from subsequent blood-feeding, were separated by 11 days. Rb was detected in ovitrap-collected eggs by atomic emission spectrophotometry. Rb-marked eggs of both species were detected up to 800 m from the release point. Eggs of Ae. albopictus were more numerous and more heterogeneously distributed in the area than those of Ae. aegypti. Eggs positively marked for Rb were found at all borders of the study area, suggesting that egg laying also occurred beyond these limits. Results from this study suggest that females can fly at least 800 m in 6 days and, if infected, potentially spread virus rapidly.     

Changes in Morphology of Trichostrongylus colubriformis Eggs and Juveniles Caused by Bacillus thuringiensis israelensis

Eggs and rhabditiform juveniles of the ruminant parasite Trichostrongylus colubriformis developed normally in Caenorhabditis briggsae Maintenance Medium. A toxin from a crystal-enriched preparation of the bacterium Bacillus thuringiensis israelensis was lethal to nematode eggs and juveniles within 24 hours and to eggs and juveniles after 24 hours of development. Treated eggs had refractive granules and development was arrested, whereas nontreated eggs developed normally. Eggs treated after 24 hours of development contained juveniles that were granulated, had esophageal derangements, and were moribund or dead. The ovicidal toxin from B. t. israelensis may facilitate microbial control of parasitic nematodes.     

Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference

The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.     

Sperm motility and fertilization time span in Atlantic salmon and brown trout—the effect of water temperature

The effect of water temperature on the duration of sperm motility, the time lapse after activation by fresh water and the fertility of eggs was studied in Atlantic salmon and brown trout. Eggs of both species were fully fertile in fresh water after 512 s. No interspecific differences were noted in egg fertility at the lower water temperatures, but the brown trout eggs showed a higher resistance to high temperatures, indicating a better physiological thermotolerance. A highly significant effect of temperature on the overall duration of sperm motility was found, with a marked peak at 3−4°C for salmon and a weaker one for trout. After freshwater activation the eggs of both species remained fertile for a longer time than the sperm were mobile.     

Biological Relationship of Rotyleinchulus borealis on Several Plant Cultivars

The embryogenic development of Rolylenchulus borealis, at 24-26 C, was completed on corn, in 12-15 days, and the life-cycle of the nematode from egg to egg required 35-40 days at 20-25 C. Juveniles remained in the soil as preinfective stages for 17-19 days before becoming adults. Only immature vermiform and swollen egg-laying females were found attached to corn roots. Eggs were laid in a gelatinous matrix on the root surface; the number of eggs per egg mass was 45 ± 28 on corn roots. Bean, green pea, potato, sorghum, and sweet potato were also found to be hosts of R. borealis. The nematode established a permanent feeding site on corn root in an endodermal cell that became hypertrophied. Pericyclic cells close to the feeding site showed granular cytoplasm and nuclei with hypertrophied nucleoli. A cell wall ingrowth was also noted around the area of stylet penetration into the endodermal cell.     

Lipovitellins and phosvitins of the fertilized eggs during embryo growth in the oviparous lizard Podarcis sicula

In the lizard Podarcis sicula, the major vitellogenin (VTG)-derived yolk proteins, lipovitellins and phosvitins, were extracted from the yolk globules of laid and fertilized eggs at different periods of incubation up to 44 days close to hatching. Embryonic development was almost over at this time. Yolk proteins were isolated by precipitation in saturated (NH(4))(2)SO(4), separated on SDS-PAGE and detected by Western blotting with homologous polyclonal anti/VTG antibody. Two lipovitellins of 110 and 116 kDa were always present in the yolk of laid eggs after 1, 10, 18, and 44 days from oviposition. Both these proteins were glycosylated and were recognized by the anti/VTG antibody; their N-terminal sequences were analyzed. Four phosvitins were detected in freshly laid eggs, but their number decreased during incubation, and after 44 days only a single protein of approximately 6.5 kDa was present. The results indicated that, in this lizard, during embryonic development, lipovitellins remain unchanged, whereas the phosphorylated components of yolk undergo continuous degradation.     

Olfactory discrimination in scat-piling lizards

Several lizard species in the Australian scincid genus Egernia havebeen reported to deposit scats in piles. We show that E. striolata,which does produce scat piles, and E. inornata, which does not,can both discriminate their own secretions, on paper substrates, fromthose of unfamiliar conspecifics. This was indicated by elevatedtongue flick rates and more time in contact with the unfamiliarstimulus. This was not just a response to a novel stimulus becausethe secretions from another species (E. stokesii) elicited lowerresponses. When scats were presented, only striolata demonstrateddiscrimination between their own scats and those of unfamiliarconspecifics. This suggests that scats could be used to produceindividual signals, perhaps indicating residence status, in scat-pilingspecies. For striolata the signal from scats became less effectiveas the scats became older, suggesting the need to pile scatsto renew the signal.     

Population Dynamics of Heterodera glycines Life Stages on Soybean

Population fluctuations of Heterodera glycines differ in fields with high and low initial population densities. In a field with low initial numbers of nematodes, the numbers of cysts and eggs in soil remained low through 100 days from planting then increased during the remainder of the growing season. In a field with high initial nematode populations, numbers increased at 30 days, decreased to low numbers at 100 days, and then resurged to maximum populations at harvest. Numbers of juveniles were greatest at 100 days in the low initial population density field and at planting in the high initial population density field. The initial numbers of eggs in the soil gave the best correlation to soil and root nematode populations 15 and 30 days later. Juveniles in the soil at planting gave the largest correlation coefficients with nematode populations in the roots at 15 days in the field with the low initial population density. Eggs and juveniles in the soil at harvest were poorly related to numbers that overwintered.     

Flow Cytometric Analysis and Sorting of Heterodera glycines Eggs

A nondestructive technique was developed to characterize and separate eggs of soybean cyst nematode, Heterodera glycines, by developmental stage using flow cytometry. Eggs from cysts cultured on susceptible soybean roots were suspended in 0.1% xanthan gum or 59% sucrose and loaded into either a Coulter EPICS 752 or EPICS 753 flow cytometer. Eggs were analyzed and sorted according to forward angle and 90° light scatter, flow cytometric parameters that are relative measures of object size and granularity, respectively. Mature eggs containing vermiform juveniles were less granular and slightly larger than eggs in earlier stages of embryogeny, allowing for separation of mature eggs from immature eggs. The effectiveness of flow cytometric sorting was evaluated by comparing the developmental stages of subpopulations of unsorted and sorted eggs. Of a subpopulation of unsorted eggs, 62% contained vermiform juveniles, whereas 85 to 95% of sorted subpopulations of larger, less granular eggs contained vermiform juveniles. Suspending H. glycines eggs in 0.1% xanthan gum or 59% sucrose for flow cytometric analysis had no effect on subsequent egg hatch in vitro. This technique is an efficient and effective means to collect large, relatively homogeneous quantities of H. glycines eggs in early or late embryogeny, and would likely be useful for analyzing and sorting eggs of other nematode species for use in developmental, genetic, or physiological research, or for identification and collection of parasitized eggs.     

The effect of transportation and confinement stress on egg production by Dicrocoelium dendriticum in sheep

The effect of transportation and confinement stress on Dicrocoelium dendriticum egg production was investigated. Sheep passing a minimum of 200 eggs g-1 of faeces were selected from a naturally infected flock. A group of six ewes (group A) was transferred to the laboratory premises and kept indoors for 28 days, while another group (B) of six ewes remained on pasture and was used as a control. Faecal examinations and egg counting were performed weekly, on all sheep, from one week before to 28 days after the transportation of the animals. Comparison of faecal egg counts between groups revealed higher (P < 0.01) counts in transported sheep sampled on days 7, 14 and 28 of the trial. Furthermore, egg counts obtained from sheep that were transferred remained consistently high while the ones from sheep that remained on pasture showed significant variation. Therefore, it is concluded that stress-inducing factors, such as transportation and confinement may enhance egg production of D. dendriticum.     

Effects of Resistance in White Clover (Trifolium repens) on Heterodera trifolii

Clones of two partially resistant and two susceptible white clover, Trifolium repens, genotypes were exposed to eggs of Heterodera trifolii and nematode development in stained roots measured at 2, 4, 7, 11, 18, 23, and 37 days after inoculation. The differences in development between nematode populations in resistant and susceptible genotypes showed that resistance operated after infection during feeding and development. At 7 days after inoculation, counts of second-stage juveniles did not differ between genotypes, whereas at 37 days more adults had developed in the susceptible than in the resistant genotypes. In a separate experiment, cysts hosted by susceptible genotypes were larger and contained more eggs than those on resistant genotypes so that the product of the values for cysts per plant and for eggs per cyst resulted in a more sensitive measure of resistance than from using cysts per plant alone.     

Comparisons of Female and Egg Assays to Identify Rotylenchulus reniformis Resistance in Cotton

More plants can be screened for reniform nematode resistance each year if the time involved can be shortened. In this study, the hypothesis that female counts are as efficient as egg counts in identifying resistant genotypes was tested. In two greenhouse experiments Gossypium genotypes which varied from resistant to susceptible to reniform nematode (Rotylenchulus reniformis) were compared to a susceptible control cultivar. Infested field soil served as the inoculum source for the first experiment, and vermiform stages extracted from greenhouse cultures were used to infest soil in the second experiment. Six replicates of each genotype were harvested 25 d after planting and swollen females were counted. The remaining plants were harvested 35 d after planting and eggs extracted from the roots were counted. Processing and counting times recorded in the first experiment were similar for both assessment methods, but 10 additional days were required for egg-based assessment. Contrast analyses showed that assessments based on females per gram of root were equivalent to assessments based on eggs per gram of root for the five genotypes tested in the first experiment and for an expanded set of 13 genotypes tested in the second experiment. The results indicated that either life stage can be used to screen for resistance.     

Discrimination of Gastrointestinal Nematode Eggs from Crude Fecal Egg Preparations by Inhibitor-Resistant Conventional and Real-Time PCR

Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles – only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM.     

Histopathology of Selected cultivars of Tobacco infected with Meloidogyne species

Rates of nematode penetration and the histopathology of root infections in fluecured tobacco cultivars ''McNair-944,'' ''Speight G-28,'' and ''NC-89'' with either Meloidogyne arenaria, M. incognita, M. hapla, or M. javanica were investigated. Penetration of root tips by juveniles of all species into the M. incognita-resistant NC-89 and G-28 was much less than that on the susceptible McNair-944. Few juveniles of M. incognita were detected in resistant cultivars 7 and 14 days after inoculation. Infection sites exhibited some cavities and extensive necrotic tissue at 14 days; less necrotic tissue and no intact nematodes were observed 35 days after inoculation. Although some females of M. arenaria reached maturity and produced eggs, considerable necrosis was induced in the resistant cultivars. Meloidogyne hapla and M. javanica developed on all cultivars, but there was necrotic tissue at some infection sites in the resistant cultivars. The occurrence of single multistructured nuclei in the syncytia of most M. hapla infections differed from the numerous small nuclei found in syncytia caused by the other three species.     

Effect of temperature, photoperiod, flooding, and drying on the hatching pattern of the eggs of Strelkovimermis spiculatus (Nematoda: Mermithidae)

We assessed the number of Strelkovimermis spiculatus preparasites obtained from a known initial number of nematode eggs and the effect of abiotic conditions (temperature, photoperiod, flooding-drying) on the number of emerged preparasites. Two egg groups were maintained: one continuously flooded, another with flooding-drying cycles (every 15, 30, 60 days). Each egg group was studied at 25°C and 14:10 (L:D) and 16°C and 12:12 (L:D). The flooded eggs contained a higher overall percentage of S. spiculatus preparasites compared to the wet-dry-cycle eggs. The conditions of continuous flooding at 16°C and 12:12 (L:D) produced the maximum percent of emerged J2s (30±15%). Preparasites were recorded by 7 (25°C) and 14 (16°C) days, suggesting this period as the minimum time for embryonic development. The preparasite-emergence time observed from the same flooded-egg batch (98 and 112 days at 25°C and 16°C, respectively) suggested a nonsynchronous hatching, possibly through nonuniform egg embryonation. The time of exposure to drought in the assays did not significantly affect the total average percentage of J2s obtained at 25°C and 14:10 (L:D), whereas at 16°C the number of emerged J2s diminished with a prolongation of the drying period. The oviposition period was also recorded only at 16°C and 12:12 (L:D): S. spiculatus eggs were detected at 12.6 days after postparasite emergence, and oviposition was complete at 51days under those conditions. We propose a flooding schedule to optimize the mass-rearing of S. spiculatus.