Development of a sterile insect technique as a control strategy for the Asian citrus psyllid: establishing the effect of sterilizing X-rays on fecundity, fertility, and survival.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a major pest of citrus due to its role as the vector of the bacterium that causes huanglongbing. In commercial citrus, ACP control currently relies on the application of insecticides, which may not be sustainable long-term, nor practical in urban areas. The sterile insect technique (SIT) is an alternative strategy in which large numbers of pests are reared, sterilized using radiation, and then released into the field to compete with wild individuals for matings, suppressing population growth. As a fundamental step toward the development of SIT for ACP, this study sought to identify the optimum radiation dose required to sterilize ACP without affecting their survival and mating capacity. Virgin adult ACP of both sexes were subjected to doses of X-ray irradiation ranging from 40 to 480 Gy, then paired with a nonirradiated mate and allowed to produce offspring. Fecundity was estimated as the number of eggs laid, and fertility as the proportion of those eggs that hatched. Females were more radio-sensitive than males, exhibiting a major drop in fecundity at even the lowest dose and 100% sterility at 80 Gy. In contrast, a fivefold higher dose (400 Gy) did not achieve complete sterility in males, with around 5% offspring survival. However, F1 progeny of males exposed to 320 Gy or higher were subsequently found to be 100% sterile. This confirmation of inherited sterility suggests that balancing the sterilizing effects of radiation against its mortality-inducing effects may warrant further evaluation.

Tracking global invasion pathways of the spongy moth (Lepidoptera: Erebidae) to the United States using stable isotopes as endogenous biomarkers.

The spread of invasive insect species causes enormous ecological damage and economic losses worldwide. A reliable method that tracks back an invaded insect's origin would be of great use to entomologists, phytopathologists, and pest managers. The spongy moth (Lymantria dispar, Linnaeus 1758) is a persistent invasive pest in the Northeastern United States and periodically causes major defoliations in temperate forests. We analyzed field-captured (Europe, Asia, United States) and laboratory-reared L. dispar specimens for their natal isotopic hydrogen and nitrogen signatures imprinted in their biological tissues (δ2H and δ15N) and compared these values to the long-term mean δ2H of regional precipitation (Global Network of Isotopes in Precipitation) and δ15N of regional plants at the capture site. We established the percentage of hydrogen-deuterium exchange for L. dispar tissue (Pex = 8.2%) using the comparative equilibration method and two-source mixing models, which allowed the extraction of the moth's natal δ2H value. We confirmed that the natal δ2H and δ15N values of our specimens are related to the environmental signatures at their geographic origins. With our regression models, we were able to isolate potentially invasive individuals and give estimations of their geographic origin. To enable the application of these methods on eggs, we established an egg-to-adult fraction factor for L. dispar (Δegg-adult = 16.3 ± 4.3‰). Our models suggested that around 25% of the field-captured spongy moths worldwide were not native in the investigated capture sites. East Asia was the most frequently identified location of probable origin. Furthermore, our data suggested that eggs found on cargo ships in the United States harbors in Alaska, California, and Louisiana most probably originated from Asian L. dispar in East Russia. These findings show that stable isotope biomarkers give a unique insight into invasive insect species pathways, and thus, can be an effective tool to monitor the spread of insect pest epidemics.

Quantifying Spillover of an Urban Invasive Vector of Plant Disease: Asian Citrus Psyllid (Diaphorina citri) in California Citrus.

Urban environments frequently play an important role in the initial stages of biological invasions, often serving as gateways for non-native species, which may propagate to nearby natural and agricultural ecosystems in the event of spillover. In California, citrus trees are a dominant ornamental and food plant in urban and peri-urban environments. We studied the invasion dynamics of the Asian citrus psyllid (Diaphorina citri), which became widespread in urban areas of southern California starting in 2008, to understand the factors driving its more recent invasion in commercial citrus groves. Using a multi-year monitoring database, we applied a suite of models to evaluate the rate at which groves accrued their first D. citri detection and the cumulative number of detections thereafter. Grove characteristics and landscape context proved to be important, with generally higher invasion rates and more cumulative detections in groves that were larger, had more edge, or had more perforated shapes, with greater urbanization intensity favoring more rapid invasion, but with inconsistent effects of distance to roads among models. Notably, distance to urban or other grove occurrences proved to be among the most important variables. During the early phase of D. citri invasion in the region, groves closer to urban occurrences were invaded more rapidly, whereas more recently, invasion rate depended primarily on proximity to grove occurrences. Yet, proximity to urban and grove occurrences contributed positively to cumulative D. citri detections, suggesting a continued influx from both sources. These results suggest that inherent features of agroecosystems and spatial coupling with urban ecosystems can be important, temporally dynamic, drivers of biological invasions. Further consideration of these issues may guide the development of strategic responses to D. citri's ongoing invasion.

Development of Sterile Insect Technique for Control of the European Grapevine Moth, Lobesia botrana, in Urban Areas of Chile.

The European grapevine moth, a Palearctic pest, was first detected in the Americas in 2008. Its establishment in Chile presented production and export issues for grapes and other fruits, and a national control campaign was launched. Urban areas next to agricultural production areas were recognized as a challenge for effective control. In 2015, a SIT laboratory was established in Arica, Chile to evaluate its potential for urban control. Progress included the development and evaluation of artificial diets, a mass-rearing of 75,000 moths/week, confirmation of 150 Gy as an operational dose for inherited sterility, and releases of sterile moths in a 25 ha urban area next to fruit production areas. Season-long releases demonstrated that high overflooding ratios were achieved early in the season but decreased with a large increase in the wild moth population. Sterile moth quality was consistently high, and moths were observed living in the field up to 10 days and dispersing up to 800 m. Recommendations for further development of the SIT include conducting cage and field studies to evaluate overflooding ratios and mating competitiveness, measuring of infestation densities in release and no-release areas, and conducting trials to evaluate combining SIT with compatible integrated pest management (IPM) tactics such as fruit stripping and use of mating disruption.

Impact of the Temperature on the Phenology of Diaphorina citri (Hemiptera: Liviidae) and on the Establishment of Tamarixia radiata (Hymenoptera: Eulophidae) in Urban Areas in the Lower Colorado Desert in Arizona.

The invasive pest Diaphorina citri Kuwayama was first detected in Arizona in 2009. Since late 2013, the parasitoid Tamarixia radiata (Waterson), the main biocontrol agent of D. citri, has been released as part of a biological control program on citrus grown in urban areas of two western Arizona counties in the lower Colorado desert environment. Here we report a 3-yr survey aimed at evaluation of T. radiata releases on D. citri populations and assess the impact of the climate conditions on the phenology of D. citri and on the establishment success of T. radiata. We also monitored the phenology of D. citri as part of this assessment on different citrus host species. We show that the high summer temperatures in the Arizona desert halt the development of D. citri for about 3 mo every year which appears to have limited the establishment and impact of T. radiata. At survey sites distant from release areas the parasitism rates over the season ranged from 0 to 75% and on average peaked around 50% in 2016 but it was low or absent in 2015 and 2017, respectively. We discuss the consequences of this phenology of D. citri in the desert areas for the prospects of long-term establishment of T. radiata and the management of this key citrus pest.

Leafroller-induced phenylacetonitrile and acetic acid attract adult Lobesia botrana in European vineyards.

We recently identified unique caterpillar-induced plant volatile compounds emitted from apple leaves infested with the larvae of various leafroller species. In subsequent field tests, binary blends of phenylacetonitrile+acetic acid and 2-phenylethanol+acetic acid were found to be attractive to a range of tortricid leafroller species (Tortricidae: Tortricinae) in both the Southern and Northern Hemispheres. In this work, the caterpillar-induced plant volatiles from the apple-leafroller system were tested in two vineyards in Spain and Hungary for their attractiveness to the grape frugivore Lobesia botrana (Tortricidae: Olethreutinae). As seen for Tortricinae species, a binary blend of phenylacetonitrile+acetic acid attracted significantly more male and female L. botrana to traps than acetic acid or blank lures. Traps baited with other caterpillar-induced plant volatile compounds (benzyl alcohol, 2-phenylethanol, indole, and (E)-nerolidol, each as a binary blend with acetic acid) did not catch significantly more moths than traps containing acetic acid alone. The catches of male and female moths support an optimistic future for new products in female tortricid surveillance and control that are based on combinations of kairomone compounds released from larval-damaged foliage.

An updated method for the jugular catheterization of grower pigs for repeated blood sampling following an oral glucose tolerance test.

Jugular catheterization is a common procedure used under experimental conditions. However, there is considerable variation in the reported techniques, particularly for grower pigs (>40 kg and <60 kg) when larger volumes of blood per sample (>10 mL) are required. This paper provides a complete methodology including the use of current equipment and anaesthetic regimen for grower pigs. This surgical jugular catheterization method was carried out in 30 large white grower pigs. Firstly, the pigs were habituated to human handling for at least two weeks prior to surgery. Animals were sedated and anesthetized. Following intubation, an incision was made in the jugular fossa, and the jugular vein was located. A catheter was then inserted and fixated. The wound was stapled and the catheter line secured to the back of the neck. The pigs recovered fully from the surgery and the catheters remained patent for the duration of the blood sampling period (min 72 h). Twenty millilitres of blood were collected every 15 min, taking approximately 2 min per pig. No haemolysis was detected in any samples. Jugular catheterization of pigs using this procedure proved successful both in terms of animal recovery and quality of samples. Catheters remained patent and pigs remained calm during sampling.

Spatiotemporal dynamics of the Southern California Asian citrus psyllid (Diaphorina citri) invasion.

Biological invasions are governed by spatial processes that tend to be distributed in non-random ways across landscapes. Characterizing the spatial and temporal heterogeneities of the introduction, establishment, and spread of non-native insect species is a key aspect of effectively managing their geographic expansion. The Asian citrus psyllid (Diaphorina citri), a vector of the bacterium associated with huanglongbing (HLB), poses a serious threat to commercial and residential citrus trees. In 2008, D. citri first began expanding northward from Mexico into parts of Southern California. Using georeferenced D. citri occurrence data from 2008-2014, we sought to better understand the extent of the geographic expansion of this invasive vector species. Our objectives were to: 1) describe the spatial and temporal distribution of D. citri in Southern California, 2) identify the locations of statistically significant D. citri hotspots, and 3) quantify the dynamics of anisotropic spread. We found clear evidence that the spatial and temporal distribution of D. citri in Southern California is non-random. Further, we identified the existence of statistically significant hotspots of D. citri occurrence and described the anisotropic dispersion across the Southern California landscape. For example, the dominant hotspot surrounding Los Angeles showed rapid and strongly asymmetric spread to the south and east. Our study demonstrates the feasibility of quantitative invasive insect risk assessment with the application of a spatial epidemiology framework.

Efficacy of Nitric Oxide Fumigation for Controlling Codling Moth in Apples.

Nitric oxide (NO) fumigation under ultralow oxygen (ULO) conditions was studied for its efficacy in controlling codling moth and effects on postharvest quality of apples. NO fumigation was effective against eggs and larvae of different sizes on artificial diet in 48 h treatments. Small larvae were more susceptible to nitric oxide than other stages at 0.5% NO concentration. There were no significant differences among life stages at 1.0% to 2.0% NO concentrations. In 24 h treatments of eggs, 3.0% NO fumigation at 2 °C achieved 100% egg mortality. Two 24 h fumigation treatments of infested apples containing medium and large larvae with 3.0% and 5.0% NO resulted in 98% and 100% mortalities respectively. Sound apples were also fumigated with 5.0% NO for 24 h at 2 °C to determine effects on apple quality. The fumigation treatment was terminated by flushing with nitrogen and had no negative impact on postharvest quality of apples as measured by firmness and color at 2 and 4 weeks after fumigation. This study demonstrated that NO fumigation was effective against codling moth and safe to apple quality, and therefore has potential to become a practical alternative to methyl bromide fumigation for control of codling moth in apples.

Oxygenated Phosphine Fumigation for Control of Light Brown Apple Moth (Lepidoptera: Tortricidae) Eggs on Cut-Flowers.

Light brown apple moth, Epiphyas postvittana (Walker), eggs were subjected to oxygenated phosphine fumigation treatments under 70% oxygen on cut flowers to determine efficacy and safety. Five cut flower species: roses, lilies, tulips, gerbera daisy, and pompon chrysanthemums, were fumigated in separate groups with 2,500 ppm phosphine for 72 h at 5°C. Egg mortality and postharvest quality of cut flowers were determined after fumigation. Egg mortalities of 99.7-100% were achieved among the cut flower species. The treatment was safe to all cut flowers except gerbera daisy. A 96-h fumigation treatment with 2,200 ppm phosphine of eggs on chrysanthemums cut flowers also did not achieve complete control of light brown apple moth eggs. A simulation of fumigation in hermetically sealed fumigation chambers with gerbera daisy showed significant accumulations of carbon dioxide and ethylene by the end of 72-h sealing. However, oxygenated phosphine fumigations with carbon dioxide and ethylene absorbents did not reduce the injury to gerbera daisy, indicating that it is likely that phosphine may directly cause the injury to gerbera daisy cut flowers. The study demonstrated that oxygenated phosphine fumigation is effective against light brown apple moth eggs. However, it may not be able to achieve the probit9 quarantine level of control and the treatment was safe to most of the cut flower species.

Oxygenated phosphine fumigation for control of Epiphyas postvittana (Lepidoptera: Tortricidae) eggs on lettuce.

Light brown apple moth, Epiphyas postvittana (Walker), is a quarantined pest in most countries. Its establishment in California and potential spread to other parts of the state and beyond make it urgent to develop effective postharvest treatments to control the pest on fresh commodities. Fumigation with cylindered phosphine at low temperature has emerged to be a practical methyl bromide alternative treatment for postharvest pest control on fresh commodities. However, its use to control E. postvittana eggs on sensitive commodities such as lettuce is problematic. E. postvittana eggs are tolerant of phosphine and long phosphine treatment also injures lettuce. In the current study, E. postvittana eggs were subjected to oxygenated phosphine fumigations to develop an effective treatment at a low storage temperature of 2 degrees C. In addition, soda lime as a CO2 absorbent was tested to determine its effects in reducing and preventing injuries to lettuce associated with phosphine fumigations. Three-day fumigation with 1,000 ppm phosphine under 60% O2 achieved 100% mortality of E. postvittana eggs in small-scale laboratory tests. In the presence of the CO2 absorbent, a 3-d large-scale fumigation of lettuce with 1,700 ppm phosphine under 60% O2 resulted in a relative egg mortality of 99.96% without any negative effect on lettuce quality. The 3-d fumigation treatment without the CO2 absorbent, however, resulted in significant injuries to lettuce and consequential quality reductions. The study demonstrated that oxygenated phosphine fumigation has the potential to control E. postvittana eggs and the CO2 absorbent has the potential to prevent injuries and quality reductions of lettuce associated with long-term oxygenated phosphine fumigation.

Effects of phosphine fumigation on survivorship of Epiphyas postvittana (Lepidoptera: Tortricidae) eggs.

Light brown apple moth, Epiphyas postvittana (Walker), eggs were subjected to phosphine fumigations under normal atmospheric and elevated oxygen levels in laboratory-scale chamber experiments to compare their susceptibilities to the two different fumigation methods. In fumigations conducted under atmospheric oxygen at 5 and 10 degrees C, egg survivorship decreased with increase in phosphine concentration but then increased at a concentration of 3,000 ppm; this increase was significant at 10 degrees C. Based on egg survivorship data, phosphine fumigations conducted in a 60% oxygen atmosphere were significantly more effective than those conducted under atmospheric oxygen conditions. Oxygenated phosphine fumigations at 5 and 10 degrees C killed all 1,998 and 2,213 E. postvittana eggs treated, respectively, after 72 h of exposure. These results indicate the great potential of oxygenated phosphine fumigation for the control of E. postvittana eggs.

Engineered repressible lethality for controlling the pink bollworm, a lepidopteran pest of cotton.

The sterile insect technique (SIT) is an environmentally friendly method of pest control in which insects are mass-produced, irradiated and released to mate with wild counterparts. SIT has been used to control major pest insects including the pink bollworm (Pectinophora gossypiella Saunders), a global pest of cotton. Transgenic technology has the potential to overcome disadvantages associated with the SIT, such as the damaging effects of radiation on released insects. A method called RIDL (Release of Insects carrying a Dominant Lethal) is designed to circumvent the need to irradiate insects before release. Premature death of insects' progeny can be engineered to provide an equivalent to sterilisation. Moreover, this trait can be suppressed by the provision of a dietary antidote. In the pink bollworm, we generated transformed strains using different DNA constructs, which showed moderate-to-100% engineered mortality. In permissive conditions, this effect was largely suppressed. Survival data on cotton in field cages indicated that field conditions increase the lethal effect. One strain, called OX3402C, showed highly penetrant and highly repressible lethality, and was tested on host plants where its larvae caused minimal damage before death. These results highlight a potentially valuable insecticide-free tool against pink bollworm, and indicate its potential for development in other lepidopteran pests.

Field performance of a genetically engineered strain of pink bollworm.

Pest insects harm crops, livestock and human health, either directly or by acting as vectors of disease. The Sterile Insect Technique (SIT)--mass-release of sterile insects to mate with, and thereby control, their wild counterparts--has been used successfully for decades to control several pest species, including pink bollworm, a lepidopteran pest of cotton. Although it has been suggested that genetic engineering of pest insects provides potential improvements, there is uncertainty regarding its impact on their field performance. Discrimination between released and wild moths caught in monitoring traps is essential for estimating wild population levels. To address concerns about the reliability of current marking methods, we developed a genetically engineered strain of pink bollworm with a heritable fluorescent marker, to improve discrimination of sterile from wild moths. Here, we report the results of field trials showing that this engineered strain performed well under field conditions. Our data show that attributes critical to SIT in the field--ability to find a mate and to initiate copulation, as well as dispersal and persistence in the release area--were comparable between the genetically engineered strain and a standard strain. To our knowledge, these represent the first open-field experiments with a genetically engineered insect. The results described here provide encouragement for the genetic control of insect pests.

Comparative fitness of irradiated light brown apple moths (Lepidoptera: Tortricidae) in a wind tunnel, hedgerow, and vineyard.

Light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), is the target of the sterile insect technique, but reduced moth fitness from irradiation lowers the effective overflooding ratio of sterile to wild moths. New measures of insect quality are being sought to improve field performance of irradiated insects, thus improving the cost effectiveness of this technique. Male pupae were irradiated at intervals between 0 and 300 Gy, and adult flight success was assessed in a wind tunnel equipped with flight track recording software. A dose response was evident with reduced successful search behaviors at higher irradiation doses. Irradiation at 250 Gy reduced arrival success to 49% of untreated controls, during 2-min assays. Mark-release-recapture of males irradiated at 250 Gy indicated reduced male moth recapture in hedgerows (75% of control values of 7.22% +/- 1.20 [SEM] males recaptured) and in vineyards (78% of control values 10.5% +/- 1.66% [SEM] recaptured). Males dispersed similar distances in both habitats, and overflooding ratios dropped off rapidly from the release point in both landscapes. Transects of traps with central releases proved to be an efficient method for measuring the quality of released males. Relative field performance of moths was greater than suggested by wind tunnel performance, which could be due to time differences between the two assays, two-minute wind tunnel tests compared with days in the field treatments. Release strategies involving ground releases should consider the effect of limited postrelease dispersal. Aerial release could solve this problem and warrants investigation.

Transport of water in small pores.

Experimental measurements of the thermal expansion coefficient (alpha), permeability (k), and diffusivity (D) of water and 1 M solutions of NaCl and CaCl(2) are interpreted with the aid of molecular dynamics (MD) simulations of water in a 3 nm gap between glass plates. MD shows that there is a layer approximately 6 A thick near the glass surface that has alpha approximately 2.3 times higher and D about an order of magnitude lower than bulk water. The measured D is approximately 5 times lower than that for bulk water. However, when the MD results are averaged over the thickness of the 3 nm gap, D is only reduced by approximately 30% relative to the bulk, so the measured reduction is attributed primarily to tortuosity of the pore space, not to the reduced mobility near the pore wall. The measured alpha can be quantitatively explained by a volume-weighted average of the properties of the high-expansion layer and the "normal" water in the middle of the pore. The permeability of the porous glass can be quantitatively predicted by the Carman-Kozeny equation, if 6 A of water near the pore wall is assumed to be immobile, which is consistent with the MD results. The properties and thickness of the surface-affected layer are not affected significantly by the presence of the dissolved salts.