Infertility and fecundity loss of Wolbachia-infected Aedes aegypti hatched from quiescent eggs is expected to alter invasion dynamics

The endosymbiotic bacterium Wolbachia shows viral blocking in its mosquito host, leading to its use in arboviral disease control. Releases with Wolbachia strains wMel and wAlbB infecting Aedes aegypti have taken place in several countries. Mosquito egg survival is a key factor influencing population persistence and this trait is also important when eggs are stored prior to releases. We therefore tested the viability of mosquitoes derived from Wolbachia wMel and wAlbB-infected as well as uninfected eggs after long-term storage under diurnal temperature cycles of 11–19°C and 22–30°C. Eggs stored at 11–19°C had higher hatch proportions than those stored at 22–30°C. Adult Wolbachia density declined when they emerged from eggs stored for longer, which was associated with incomplete cytoplasmic incompatibility (CI) when wMel-infected males were crossed with uninfected females. Females from stored eggs at both temperatures continued to show perfect maternal transmission of Wolbachia, but storage reduced the fecundity of both wMel and wAlbB-infected females relative to uninfected mosquitoes. Furthermore, we found a very strong negative impact of the wAlbB infection on the fertility of females stored at 22–30°C, with almost 80% of females hatching after 11 weeks of storage being infertile. Our findings provide guidance for storing Wolbachia-infected A. aegypti eggs to ensure high fitness adult mosquitoes for release. Importantly, they also highlight the likely impact of egg quiescence on the population dynamics of Wolbachia-infected populations in the field, and the potential for Wolbachia to suppress mosquito populations through cumulative fitness costs across warm and dry periods, with expected effects on dengue transmission.     

Effects of Initial Nematode Density on Population Dynamics of Globodera rostochiensis on Resistant and Susceptible Potatoes

The influence of resistant and susceptible potato cultivars on Globodera rostochiensis population density changes was studied at different nematode inoculum levels (Pi) in the greenhouse and field. Soil in which one susceptible and two resistant cultivars were grown and fallow soil in pots was infested with cysts to result in densities of 0.04-75 eggs/cm³ soil. A resistant cultivar was grown in an infested field with Pi of 0.7-16.7 eggs/cm³ soil. Pi was positively correlated with decline of soil population densities due to hatch where resistant potatoes were grown in the greenhouse and in the field but not in fallow soil. However, Pi was not correlated with in vitro hatch of G. rostochiensis cysts in water or potato root diffusate. Under continuous culture o f a resistant cultivar, viable eggs per cyst declined 60-90% per plant growth cycle (4 weeks) and the number of cysts containing viable eggs had decreased by 77% after five cycles. The rate of G. rostochiensis reproduction on both resistant and susceptible cultivars was negatively correlated with Pi. These data were used to predict the effect of resistant and susceptible potato cultivars on G. rostochiensis soil population dynamics.     

The Relationship between Temperature and Development in Globodera ellingtonae

A new cyst nematode species, Globodera ellingtonae, was recently described from populations in Oregon and Idaho. This nematode has been shown to reproduce on potato. Because of this nematode’s close relationship to the potato cyst nematodes, G. rostochiensis and G. pallida, an understanding of the risk of its potential spread, including prediction of potential geographical distribution, is required. To determine the development of G. ellingtonae under different temperatures, we conducted growth chamber experiments over a range of temperatures (10.0°C to 26.5°C) and tracked length of time to various developmental stages, including adult females bearing the next generation of eggs. Both the time to peak population densities of G. ellingtonae life stages and their duration in roots generally increased with decreasing temperature. Regression of growth rate to second-stage (J2) and third-stage (J3) juveniles on temperature yielded different base temperatures: 6.3°C and 4.4°C for J2 and J3, respectively. Setting a base temperature of 6°C allowed calculation of the degree-days (DD6) over which different life stages occurred. The largest population densities of J2 were found in roots between 50 and 200 DD6. Population densities of J3 peaked between 200 and 300 DD6. Adult males were detected in soil starting at 300 to 400 DD6 and remained detectable for approximately 500 DD6. By 784 to 884 DD6, half of the eggs in adult females contained vermiform juveniles. Given the similarity in temperature ranges for successful development between G. ellingtonae and G. rostochiensis, G. ellingtonae populations likely could survive in the same geographic range in which G. rostochiensis now occurs.     

Host Status of Different Potato (Solanum tuberosum) Varieties and Hatching in Root Diffusates of Globodera ellingtonae

Globodera ellingtonae was detected in Oregon in 2008. In order to make decisions regarding the regulation of this nematode, knowledge of its biology is required. We determined the host status of a diversity of potato (Solanum tuberosum) varieties in soil-based experiments and identified hatching stimulants in in vitro hatching assays. ‘Russet Burbank,’ ‘Desiree,’ ‘Modac,’ ‘Norland,’ ‘Umatilla,’ and ‘Yukon Gold’ were good hosts (RF > 14) for G. ellingtonae. Potato varieties ‘Maris Piper,’ ‘Atlantic,’ and ‘Satina,’ all which contain the Ro1 gene that confers resistance to G. rostochiensis, were not hosts for G. ellingtonae. In in vitro hatching assays, G. ellingtonae hatched readily in the presence of diffusates from potato (PRD) and tomato (Solanum lycopersicum; TRD). Egg hatch occurred in an average of between 87% and 90% of exposed cysts, with an average of between 144 and 164 juveniles emerging per cyst, from PRD- and TRD-treated cysts, respectively. This nematode hatched rapidly in the presence of PRD and TRD, with at least 66% of total hatch occurring by day 3 of exposure. There was no dose-response of egg hatch to concentrations of PRD or TRD ranging from 1:5 to 1:100 diffusate to water. When G. ellingtonae was exposed to root diffusates from 21 different plants, hatch occurred in 0% to 70% of exposed cysts, with an average of between 0 to 27 juveniles emerging per cyst. When root diffusate-exposed cysts were subsequently transferred to PRD to test viability, root diffusates from arugula (Eruca sativa), sudangrass (Sorghum bicolor subsp. drummondii), and common vetch (Vicia sativa) continued to inhibit egg hatch compared with the other root diffusates or water in which hatch occurred readily (60 to 182 juveniles emerging per cyst). Previously known hatching stimulants of G. rostochiensis and G. pallida, sodium metavanadate, sodium orthovanadate, and sodium thiocyanate, stimulated some egg hatch. Although, Globodera ellingtonae hatched readily in PRD and TRD and reproduced on potato, the pathogenicity of this nematode on potato remains to be determined.     

Evaluation of Dry Ice as a Potential Cryonematicide for Meloidogyne incognita in Soil

Solid CO₂ (dry ice) was added to pots containing soil that was infested either with eggs of the root-knot nematode, Meloidogyne incognita, or with tomato (Lycopersicon esculentum ''Rutgers'') root fragments that were infected with various stages of the nematode. Two hours after dry ice was added, thermocouples in the soil recorded temperatures ranging from -15 °C to -59 °C. One day after treatment with the dry ice, the temperature of the soil was allowed to equilibrate with that of the greenhouse, and susceptible tomato seedlings were planted in pots containing infested soil treated or untreated (controls) with dry ice. After 5 weeks, roots were removed from the pots and nematode eggs were extracted and counted. Plants grown in soil infested with eggs and receiving dry ice treatment had less than 1% of the eggs found in the controls; plants from soil infested with root fragments and receiving dry ice treatment had less than 4% of the eggs found in controls. Dry ice used to lower soil temperature may have potential as a cryonematicide.     

Degree-day Models for Predicting Egg Hatch and Population Increase of Criconemella xenoplax

A degree-day model was derived to predict egg hatch for Criconemella xenoplax. Eggs collected from gravid females were incubated in distilled water at constant temperatures of 10-35 C. Sixty-six percent of all eggs hatched between 13 and 32 C, and 42% hatched at 10 C. All eggs aborted above 32.5 C. Between 25 and 32 C, 8.5 ± 0.5 days were required for egg hatch. Degree-day requirement for egg hatch at 10-30 C was estimated to be 154 ± 5 with a base of 9.03 ± 0.04 C. This base of 9 C was adopted in studies of the relationship between degree-days and nematode population increase on Prunus seedlings grown 9-11 weeks in a greenhouse. Degree-day accumulations were based upon daily averages from maximum and minimum air temperatures. Ratios of final to initial population densities exhibited an exponential pattern in relation to degree-day accumulations with proportionate doubling increment of 0.100 ± 0.049 every 139 ± 8 degree-days. These results provide a means of predicting nematode population increase under greenhouse conditions and a basis for choosing sampling intervals when evaluating nematode multiplication.     

Hatching of Meloidogyne incognita Eggs in the Neutral Carbohydrate Fraction of Root Exudates of Gnotobiotically Grown Alfalfa

Meloidogyne incognita eggs were hatched in soil sterilized by gamma kradiation and wetted with root exudates from alfalfa plants in different stages of development and subjected to various levels of clipping. Carbohydrate components of the exudates were identified by gas chromatography-mass spectrometry. Although significant stimulation of hatch was detected in exudates of seedling and flowering plants, the practical importance of the increase is doubtful as hatch in distilled water was always greater than 50%. Hatch did not differ among exudate samples from clipped plants. Incubation of eggs in soil moistened with 10⁻⁷ to 10⁻³ M solutions of glucose did not result in increased hatching over that in distilled water.     

TIME-TEMPERATURE RELATIONS IN THE INCUBATION OF THE WHITEFISH,COREGONUS CLUPEAFORMIS (MITCHILL)

1. Whitefish eggs incubated in aerated lake water at controlled tempera tures of 0°, 0.5°, 2°, 4°, 6°, 8°, 10°, and 12°C., failed to hatch at either 0° or 12°C. 0.6 per cent hatched alive at 10°C., 72.67 per cent hatched alive at 0.5°C., and an intermediate proportion hatched at intermediate temperatures. 2. The percentage of abnormal embryos which developed to the hatching stage varied directly with temperature between 4° and 12°, all embryos being abnormal at 12°C.; but none were abnormal at either 0.5°, or 2°C. Normal development predominated from 0.5 to 6°C. The highest proportion of embryos to hatch alive was 72.67 per cent at 0.5°C., which is, hence, the optimum temperature. 3. Total incubation time ranged from 29.6 days at 10°C. to 141 days at 0.5°C. 4. The time (T) required to attain any given stage of development is expressed in equations See PDF for Equation where temperature, t, is a negative exponent of the constant, A, whose value differs above or below 6°C., a critical temperature. Values of A above 6° fluctuate about 1.13; those of A below 6° fluctuate about 1.19 as a mean. 5. Applying Arrhenius'' equation µ values for the total incubation period are 27,500 below 6° and 27,100 above it. 6. The relative magnitude of A values of the exponential equation and µ values of Arrhenius'' equation show corresponding changes from one developmental period to another. 7. When plotted, thermal increments show cyclic variations, with maxima during periods of cleavage and of organogenesis. These may indicate the interaction of two separate sets of embryonic processes, which give a maximal response to temperature differences during these two separate periods. 8. Above 6°, µ values during the hatching process are distinct from those of developmental stages and are regarded as being due to the action of hatching enzymes.     

Influence of Temperature on Multiplication and Egg Hatching of Longidorus africanus

Longidorus africanus multiplication on tomato was highest at 29 °C. Few nematodes were recovered after 6 weeks at soil temperatures of 35 °C or below 23 °C. The time to egg hatching was shortest and the percentage of eggs hatching was highest at 29 °C. The minimum temperature and the heat sum above this temperature required for egg development were calculated to be 14.3 °C and 94.08 degree-days, respectively. The thermal times required for egg development by L. africanus and L. elongatus were nearly identical. For both species the product of the base temperature and the heat sum was near constant, and at a temperature of 22.3 °C the rates of egg development were equal.     

Factors Affecting Egg Hatch of Heterodera mediterranea and Differential Responses of Olive Cultivars to Infestation

The influence of temperature and olive root exudates on Heterodera mediterranea egg hatch and the effects of H. mediterranea on the growth of two olive cultivars (Arbequina and Picual) were investigated. Egg hatch occurred over a temperature range of 10 to 30°C and was optimal at 20 to 25°C. There were no differences in egg hatch between sterile deionized distilled water or root exudate dilutions (undiluted, diluted 1:1, and 1:2) of Arbequina and Picual at 20°C. Heterodera mediterranea reproduced on both olive cultivars in growth chambers at 25°C. Soil and root final nematode populations, as well as total number of cysts per plant and reproduction rate, were significantly higher in Arbequina than in Picual. Shoot dry and root fresh weights as well as increases of shoot height, trunk diameter, and numbers of nodes were significantly suppressed by infection with 10,000 eggs + second-stage juveniles/pot in Arbequina but not in Picual.     

Observations on periodicity of hatching of eggs of the potato cyst nematode, Heterodera rostochiensis Woll.

Soil containing new-generation cysts of Heterodera rostochiensis was taken from the field at monthly intervals during late summer and autumn and kept in various conditions for up to a year. The number of eggs that hatched in the stored cysts was compared each month with the number that hatched in cysts taken directly from the field. Eggs did not hatch readily when stimulated during the late autumn and early winter, although more did so in cysts taken from the field before August than after. A few more eggs hatched in cysts stored in air-dried soil than in cysts stored in moist soil. Some cysts were kept at 15 or 20 °C continuously and others at 5, 15 or 30 °C for 6 weeks followed by 20 °C continuously. Storage at 30 °C caused eggs to hatch sooner, but otherwise the temperature of storage had little effect on hatch at any time of the year. Warmth also increased the hatch of H. cruciferae sooner, and some synthetic hatching agents did so with both of these species. When freed from new cysts, more eggs of H. rostochiensis hatched than in intact cysts and hatch was further increased when the fragments of tanned cyst-wall were left with the freed eggs. Puncturing the cyst-wall of new brown cysts had little effect on the hatch in potato root diffusate. Like eggs in new cysts, those in 1-year-old cysts stored out of doors ceased to hatch during the autumn and winter. The term ‘dormancy’ is inadequate to describe the inability of eggs of H. rostochiensis and other Heterodera spp. to hatch in the appropriate stimulant and the term ‘facultative diapause’, as applied to insects, better fits the phenomenon.     

Laboratory studies of factors affecting egg hatch of triops longicaudatus (LeConte) (Notostraca : Triopsidae)

Egg hatch was greatest (78.33%) for eggs not previously desiccated. A reduction in numbers hatched occurred as the relative humidity at which they were dried decreased. Some eggs hatched (0.67–79.33%) at pH levels of 3.10–10.01 with the highest hatch at pH 5.60. Water temperature greatly affected egg hatch. No hatch occurred until temperatures were above 14°C. A constant 29°C significantly inhibited hatching. Egg hatch increased 13.00 to 43.42% as salinity decreased from 2200 to 9.24 micromhos/cm. As little as 13 mm of flooded soil covering the eggs prevented them from hatching for 14 days. Eighteen percent hatch resulted when soil and eggs were redistributed to a 1 mm soil layer. Egg samples from the same parent, even though treated similarly, often hatched at greatly varying rates and only rarely was hatching 100% within a replication.     

Effect of Temperature on Pratylenchus penetrans Development

Reproduction and development of Pratylenchus penetrans were studied on genetically transformed ladino clover roots. Solitary females developing on transformed roots in nutrient gellan gum medium (pH 5.5) deposited 1.2, 1.5, 1.6, 1.8, and 2.0 eggs per day at the respective temperatures of 17, 20, 25, 27, and 30 °C. The number of eggs deposited was highly correlated with temperature. A reduction in egg-laying rates at the start of hatching was observed at all temperatures. Juvenile mortality was higher at 17 °C (50.4%), 20 °C (50.3%), and 30 °C (58.4%) than at 25 °C (34.6%) and 27 °C (37.6%). Life-cycle (egg deposition to egg deposition) duration was 46, 38, 28, 26, and 22 days at the respective temperatures. The developmental zero degrees (°C) and the effective accumulative temperatures (degree-days) required for hatching, female emergence, and onset of oviposition (completion of one generation) of P. penetrans were estimated to be 2.7 and 200, 4.2 and 548, and 5.1 and 564, respectively. Pratylenchus penetrans reproduces over a wide range of temperatures.     

Differences in Hatching of Heterodera glycines Egg-mass and Encysted Eggs in vitro

Hatching studies with Heterodera glycines typically have been conducted with a mixture of egg-mass and encysted eggs. Laboratory research was conducted to compare hatching of H. glycines eggs from external egg masses with that of eggs extracted from within females and cysts (encysted eggs). Egg-mass eggs were collected by soaking infected soybean roots in 0.5% sodium hypochlorite, and encysted eggs were collected from females and cysts dislodged from the same roots with a stream of water. Eggs were incubated at 25 °C in deionized water, 3.0 mM ZnSO₄solution, or one of three synthetic H. glycines hatch inhibitors, mad hatched juveniles were counted every other day for 22 days. Samples of eggs collected at the beginning and end of all experiments were analyzed to determine extent of embryo development. Egg-mass eggs hatched more rapidly than encysted eggs during the first 16 days, but not thereafter. Throughout the experiments, hatch of egg-mass eggs in deionized water was greater than that of encysted eggs. From day 8 to day 22, egg-mass eggs were less sensitive than encysted eggs to the hatch inhibitor 2-(2''-carboxyethyl)-5-[carboxy(hydroxy)methylidenyl]cyclopentanone. A greater proportion of egg-mass eggs contained vermiform juveniles than did encysted eggs at the beginning of the experiments, but not at the end. Results indicated that H. glycines egg-mass and encysted eggs have different hatching behaviors that cannot be explained entirely by differences in embryological development.     

Exposure Time to Lethal Temperatures for Meloidogyne incognita Suppression and Its Implication for Soil Solarization

Meloidogyne incognita eggs or J2 were incubated in test tubes containing sand:peat mix and immersed in a water bath heated to 38, 39, 40, 41, 42, 43, 44 and 45°C for a series of time intervals. Controls were maintained at 22°C. Nematodes surviving or hatching were collected from Baermann trays after three weeks of incubation. Regression analyses between percent survival or egg hatch and hours of heat treatment were performed for each temperature. Complete suppression of egg hatch required 389.8, 164.5, 32.9, 19.7 and 13.1 hours at 38, 39, 40, 41 and 42°C, respectively. Complete killing of J2 required 47.9, 46.2, 17.5 and 13.8 hours at 39, 40, 41 and 42°C, respectively. J2 were not completely killed at 38°C within 40 hours of treatment, but were killed within one hour at 44 and 45°C. Effect of temperature on nematode killing is not determined by heat units. Oscillating temperature between cool and warm did not interfere with the nematode suppressive effect by the heat treatment. Six-week solarization in the field during the summers of 2003 and 2004 in Florida accumulated heat exposure times in the top 15 cm of soil that surpassed levels required to kill M. incognita as determined in the water bath experiments. Although near zero M. incognita were detected right after solarization, the nematode population densities increased after a cycle of a susceptible pepper crop. Therefore, future research should address failure of solarization to kill nematodes in the deeper soil layers.     

Reproduction of Heterodera schachtii Schmidt on Resistant Mustard,Radish, and Sugar Beet Cultivars

The reproduction of a Wyoming population of Heterodera schachtii was determined for resistant trap crop radish (Raphanus sativus) and mustard (Sinapis alba) cultivars, and resistant and susceptible sugar beet (Beta vulgaris) cultivars in a greenhouse (21 °C/16 °C) and a growth chamber study (25 °C). Oil radish cultivars also were field tested in 2000 and 2001. In the greenhouse study, reproduction was suppressed similarly by the resistant sugar beet cultivar Nematop and all trap crop cultivars (P ≤ 0.05). In the growth chamber study, the radish cultivars were superior to most of the mustard cultivars in reducing nematode populations. All trap crops showed less reproduction than Nematop (P ≤ 0.05). In both studies, Nematop and all trap crops had lower Pf than susceptible sugar beet cultivars HH50 and HM9155 (P ≤ 0.05). In field studies, Rf values of radish cultivars decreased with increasing Pi of H. schachtii (r² = 0.59 in 2000 and r² = 0.26 in 2001). In 2000, trap crop radish cv. Colonel (Rf = 0.89) reduced nematode populations more than cv. Adagio (Rf = 4.67) and cv. Rimbo (Rf = 13.23) (P ≤ 0.05) when Pi was lower than 2.5 H. schachtii eggs and J2/cm³ soil. There were no differences in reproductive factors for radish cultivars in 2001 (P ≤ 0.05); Rf ranged from 0.23 for Adagio to 1.31 for Commodore for all Pi.     

Mineral Soils as Carriers for Rhizobium Inoculants

Mineral soil-based inoculants of Rhizobium meliloti and Rhizobium phaseoli survived better at 4°C than at higher temperatures, but ca. 15% of the cells were viable at 37°C after 27 days. Soil-based inoculants of R. meliloti, R. phaseoli, Rhizobium japonicum, and a cowpea Rhizobium sp. applied to seeds of their host legumes also survived better at low temperatures, but the percent survival of such inoculants was higher than peat-based inoculants at 35°C. Survival of R. phaseoli, R. japonicum, and cowpea rhizobia was not markedly improved when the cells were suspended in sugar solutions before drying them in soil. Nodulation was abundant on Phaseolus vulgaris derived from seeds that had been coated with a soil-based inoculant and stored for 165 days at 25°C. The increase in yield and nitrogen content of Phaseolus angularis grown in the greenhouse was the same with soil-and peat-based inoculants. We suggest that certain mineral soils can be useful and readily available carriers for legume inoculants containing desiccation-resistant Rhizobium strains.     

Survival of Cowpea Rhizobia in Soil as Affected by Soil Temperature and Moisture

Successful inoculation of peanuts and cowpeas depends on the survival of rhizobia in soils which fluctuate between wide temperature and moisture extremes. Survival of two cowpea rhizobial strains (TAL309 and 3281) and two peanut rhizobial strains (T-1 and 201) was measured in two soils under three moisture conditions (air-dry, moist (−0.33 bar), and saturated soil) and at two temperatures (25 and 35°C) when soil was not sterilized and at 40°C when soil was sterilized. Populations of rhizobia were measured periodically for 45 days. The results in nonsterilized soil indicated that strain 201 survived relatively well under all environmental conditions. The 35°C temperature in conjunction with the air-dry or saturated soil was the most detrimental to survival. At this temperature, the numbers of strains T-1, TAL309, and 3281 decreased about 2 logs in dry soil and 2.5 logs in saturated soil during 45 days of incubation. In sterilized soil, the populations of all strains in moist soil increased during the first 2 weeks, but decreased rapidly when incubated under dry conditions. The populations did not decline under saturated soil conditions. From these results it appears that rhizobial strains to be used for inoculant production should be screened under simulated field conditions for enhanced survival before their selection for commercial inoculant production.     

Diapause and prolonged development in the embryo and their ecological significance in two cicadas, Cryptotympana facialis and Graptopsaltria nigrofuscata

The seasonal timing mechanism of egg hatching was examined in two cicadas, Cryptotympana facialis and Graptopsaltria nigrofuscata, with different but overlapping geographical distributions. These species lay eggs in summer, and nymphs hatch in the summer of the following year after egg durations of 10-12 months. When eggs were maintained at 25 °C from oviposition, both the species entered embryonic diapause within 60 days irrespective of photoperiod, but at different developmental stages between the two species. The optimal temperature for diapause development was approximately 15 °C in both the species. The development rate for postdiapause morphogenesis increased linearly with temperature in the range of 20-27.5 °C in C. facialis, and of 15-25 °C in G. nigrofuscata. The lower development threshold and the sum of effective temperatures were computed as 14.3 °C and 715.3 day-degrees in C. facialis and 12.1 °C and 566.6 day-degrees in G. nigrofuscata, respectively. The hatching dates predicted by these large thermal constants accorded with the hatching dates observed in the field, i.e., late June and mid-July in G. nigrofuscata and C. facialis, respectively. Therefore, the high thermal requirements for postdiapause development compel the cicadas to hatch in summer.     

Effect of Artemisia vulgaris Rhizome Extracts on Hatching,Mortality, and Plant Infectivity of Meloidogyne megadora

The activity of an ethanolic rhizome extract of Artemisia vulgaris against hatching, mortality, host plant infectivity, and galling of the root-knot nematode Meloidogyne megadora was investigated. The extract inhibited egg hatch (50% inhibition by 2.35mg/ml) and caused second-stage juvenile mortality (50% lethality at 12 hours'' exposure to 55.67 mg/ml), both in a dose-dependent manner. Nematode infectivity on Phaseolus vulgaris ''Bencanta Trepar'', a susceptible host, decreased in a dose-responsive manner (50% inhibition at 6.28 hours exposure to extract). When applied directly to the soil, the extract reduced root galling on a susceptible host in a dose-dependent manner (50% inhibition by 32.36 mg/ml). After dilution in distilled water, the extract did not lose activity when stored in the dark at 25°C for 15 days.