The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.     

Photoperiodic and Thermal Regulation of Development and Cold Hardiness in Larvae of the Clover Leaf Weevil, Hypera punctata

Effects of photoperiod and temperature on the development and cold hardiness were investigated in larvae of Hypera punctata. At a relatively low temperature (15°C), the larvae fed less and developed more slowly under a 12L:12D (SD) photoperiod than under a 16L:8D photoperiod (LD). SD larvae had lower gut weight against the whole body weight and lower supercooling point (SCP) than the LD counterparts for the same instar and same body weight. This was because the larval SCP is markedly affected by the quantity of the gut content. Laboratory experiments indicated that the low temperature mortality of this larvae occurred mainly due to freezing irrespective of the photoperiod and temperature, suggesting that the lower lethal temperature (LLT) depends on the supercooling ability of larvae. The SD larvae tended to have a lower SCP and hence a lower LLT than the LD counterparts at 15 or 10°C, unlike at 20°C. Thus, the slower larval development under SD conditions at relatively low temperatures may prevent larvae from reaching the later instar, which have a higher SCP and thus less cold tolerance, during the coldest season. The suppressed feeding activity under SD conditions would lower the SCP, thereby reducing the possibility of lethal tissue freezing. Such a photoperiodic and thermal regulation of the larval development and the supercooling ability appear to represent adaptive mechanisms for winter survival in this beetle.     

The Ability of Coptera haywardi (Ogloblin) (Hymenoptera: Diapriidae) to Locate and Attack the Pupae of the Mediterranean Fruit Fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), under Seminatural Conditions

In Latin America, the diapriid Coptera haywardi (Ogloblin) attacks the pupae of tephritid fruit flies. Anastrepha spp. are among its natural hosts, but in the laboratory it also develops in the exotic Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Field cage tests demonstrated that C. haywardi could locate and parasitize Mediterranean fruit fly pupae under seminatural conditions as found in a Guatemalan coffee plantation. A mean of 18.3% of the pupae buried artificially at depths of 5 mm were parasitized by C. haywardi, while those buried at 15 mm suffered 3.2% parasitism. In a laboratory experiment, larvae that buried themselves to pupate were not significantly more likely to be parasitized than artificially buried pupae, although they may have left a physical or chemical trail that betrayed their presence. Thus, the artificial burial of pupae is unlikely to grossly underestimate C. haywardi efficacy in the field. Another field cage test found that mortality levels due to unsuccessful parasitoid attacks were similar to those resulting from successful parasitism. Thus, the actual effect of a mass-release might be considerably greater than that suggested from parasitism data alone. The results are considered sufficiently positive to encourage further testing of C. haywardi as a biological control agent of the Mediterranean fruit fly.     

Interactions of two natural enemies of Tetranychus evansi, the fungal pathogen Neozygites floridana (Zygomycetes: Entomophthorales) and the predatory mite, Phytoseiulus longipes (Acari: Phytoseiidae)

Tetranychus evansi is an exotic pest of Solanaceous crops in Africa discovered in Zimbabwe in 1979. Two natural enemies, the predatory mite Phytoseiulus longipes and the fungal pathogen Neozygites floridana are important causes of mortality in T. evansi populations in Brazil. The first part of this study assessed the effects of N. floridana on predation and oviposition of P. longipes fed on N. floridana infected T. evansi and T. urticae. No N. floridana hyphal bodies were found in P. longipes after this feeding, demonstrating that N. floridana is not pathogenic to P. longipes and does not affect its oviposition. The second part of the study investigated the time spent on searching for and consuming of eggs on leaf discs with and without N. floridana capilliconidia. Both the searching and the feeding time on the first egg were similar on leaf discs with and without capilliconidia. When P. longipes was offered the choice of feeding on eggs on leaf discs with or without capilliconidia, the numbers of eggs consumed were not different. The only N. floridana effect observed on P. longipes was reduced egg predation. In addition, increased time spent grooming on leaf discs with capilliconidia was observed. P. longipes was efficient in removing most capilliconidia attached to the body through self-grooming behavior. This suggests that although the predator did not avoid areas with capilliconidia, it detected and removed most capilliconidia attached to the body. Increased grooming may account for the lower egg predation rates.     

Biological control of an exotic scale, Orthezia insignis Browne (Homoptera: Ortheziidae), saves the endemic gumwood tree, Commidendrum robustum (Roxb.) DC. (Asteraceae) on the island of St. Helena

St. Helena is a small South Atlantic island with a highly degraded, but internationally significant, terrestrial flora. The 2500 remaining gumwoods, Commidendrum robustum, are an important part of this remnant flora. In 1991, a South American scale insect, Orthezia insignis, was identified attacking the gumwood trees. By 1993, severe infestations had killed over 100 trees. If the number of dead trees had continued to increase exponentially, all 2500 trees would have died by 1995. This outcome was likely given abundant alternative host plants, and lack of natural enemies of O. insignis. Host range tests showed that the other members of the endemic genus Commidendrum were also at risk. The coccinellid predator, Hyperaspis pantherina, was released on St. Helena in 1993. H. pantherina numbers increased from 1994, coinciding with a 30× decrease in scale numbers. Scale outbreaks have not been reported on St Helena since 1995, and culturing of H. pantherina was discontinued because insufficient O. insignis prey could be found in the field. Extensive blackening from sooty molds on the surviving trees in 1995 suggested that the predator was effective just in time to prevent death of most trees. H. pantherina appears to have saved the field population of a rare endemic plant from extinction.     

Field Releases of the Predatory Mite Neoseiulus fallacis (Acari: Phytoseiidae) in Canada, Monitored by Pyrethroid Resistance and Allozyme Markers

The predacious phytoseiid mite Neoseiulus fallacis (Garman) is an important agent for the biological control of spider mites in deciduous fruit orchards in North America and Canada. It would be helpful to monitor the fate of released individuals to improve the results of introductions of the predators in biological control trials. We have used two types of genetic markers, pyrethroid resistance and allozymes, for indirect estimation of the survival of N. fallacis introduced in an apple orchard in Ontario, Canada. Mite samples were submitted to toxicological tests. The polymorphism of four enzymes was studied in individual females using an isoelectric focusing technique. A mite sample was taken from the field, mass-reared in the laboratory, and selected for permethrin resistance. This strain was released on several apple trees treated with permethrin, and mite samples were collected from the same trees 10 to 90 days later. The genetic composition and the insecticide resistance level of this sample were compared to those of two other samples from trees where mites had not previously been released, either in the same orchard or in a neighboring block. A control susceptible strain was compared using mites collected earlier from trees on the same site but outside the present experiment. The mites collected from the release trees and those from the strain used for the releases were found to be genetically closely related, as judged from a small genetic distance, and from similar levels of insecticide resistance in both samples. The control samples from the nonrelease trees were genetically distant from these and displayed low resistance levels. These results indicate that the released genotypes established and persisted in the release trees for the period of the experiment. The utility of the two approaches in assessing the fate of released natural enemies is discussed.     

Host Specificity of the Argentine Root-Boring Weevil, Heilipodus ventralis (Coleoptera: Curculionidae), a Potential Biocontrol Agent for Snakeweeds (Gutierrezia: Asteraceae) in Western North American Rangelands—U.S. Quarantine Tests

Native snakeweeds, especially Gutierrezia sarothrae (Pursh) Britton and Rusby and Gutierrezia microcephala (DC.) A. Gray, are among the most widespread and damaging weeds of rangelands in the western United States and northern Mexico. The genus long ago spread to southern South America, where further speciation occurred. We have found several species of insects in Argentina that damage other species of snakeweeds there and are possible candidates for biological control in North America. The first of these, the root-boring weevil, Heilipodus ventralis (Hustache), was tested in Argentina and then sent to the USDA-ARS Insect Quarantine Facility at Temple, Texas, for host specificity testing on North American plants. We tested H. ventralis on 40 species of the family Asteraceae, in 19 tests of five types, using 686 adults and 365 larvae. Host specificity increased from adult feeding, to ovipositional selection, to larval development. At Temple, adults fed mostly on 6 species of the closely related genera Grindelia, Gutierrezia, and Gymnosperma, but with substantial feeding on four other genera of the two preferred subtribes Solidagininae and Machaerantherinae and on Baccharis in the tribe Baccharidinae, with lesser feeding on the subtribe Asterinae, all in the tribe Astereae, and on 1 species in the tribe Anthemideae. Females oviposited primarily on the same 6 species but very little on plants outside the 2 preferred subtribes. Larvae developed only on 9 of the 29 U.S. plant species tested, 6 within the two preferred subtribes and on Brickellia and Aster in other tribes. Only 5 species of three genera appear to be potential true hosts of H. ventralis in North America, on which all stages of the life cycle, adult feeding, oviposition, and larval development, can take place; these are Gymnosperma glutinosum (Spreng.) Less., Gutierrezia grandis Blake, Gut. microcephala, Gut. sarothrae, and Grindelia lanceolata Nutt. None of these genera contain species of economic or notable ecological value; the few rare species appear to be protected by habitat isolation from attack by H. ventralis. H. ventralis, therefore, appears sufficiently host specific for field release in North America. This is the first introduced biocontrol agent to be approved for release in a continental area to control a native weed.     

The effect of larval predators Thanasimus dubius (Coleoptera: Cleridae), produced by an improved system of rearing, against the southern pine beetle Dendroctonus frontalis (Coleoptera: Curculionidae)

The southern pine beetle, Dendroctonus frontalis (Coleoptera: Curculionidae) (SPB), is known to be a major bark beetle pest of pines throughout the southeastern United States. A common predator of bark beetles, Thanasimus dubius (Coleoptera: Cleridae), has been suggested to play a prevalent role on SPB dynamics. Evaluations of T. dubius have been limited by rearing methods; an artificial diet for larval T. dubius exists, and preservatives such as sorbic acid could help to maximize diet shelf-life and enhance the efficiency of the rearing system. The effects of sorbic acid at different concentrations (0%, 0.1% and 0.2%) in the larval diet for T. dubius were measured, and the effects of increased feeding time intervals (2-3 vs. 5 days) on predator performance evaluated. In addition, an experimental bioassay was conducted where newly hatched T. dubius larvae were released at four densities (0, 50, 100, and 200 per log) on pine logs infested by SPB. Sorbic acid in the diet reduced female fecundity (by 20-40%), but did not affect adult T. dubius size or longevity. However, using this preservative may not be necessary because it had no effect on the overall efficiency of the rearing system, while refreshing the larval diet every 5 days (compared with 2-3 days) did improve its efficiency, even without sorbic acid. The release of larval T. dubius resulted in a highly significant effect on the SPB ratio of increase (RI). This experiment was facilitated by the improvements in our rearing methods.     

Co-induction of DNA relaxation and synthesis of DnaK and GroEL proteins inEscherichia coli by expression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor

We examined the influence of overexpression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor ofEscherichia coli, on DNA supercoiling and induction of heat shock proteins. Cells were transformed with a plasmid carrying the structural gene for LetD protein under control of thetac promoter, and LetD protein was induced by adding isopropyl-d-thiogalactopyranoside (IPTG) to the culture medium. Analysis by agarose gel electrophoresis in the presence of chloroquine revealed relaxation of plasmid DNA in cells depending on the concentration of IPTG employed for induction. Protein pulse-labeling experiments with [³⁵S]methionine and cysteine revealed that synthesis of DnaK and GroEL proteins was also induced by IPTG, and concentrations necessary for DNA relaxation and induction of the heat shock proteins were much the same. Expression of mutant LetD protein lacking two amino acid residues at the C-terminus induced neither DNA relaxation nor the synthesis of DnaK and GroEL proteins. Induction of wild-type LetD protein but not mutant LetD protein markedly enhanced synthesis of ³². We interpret these results to mean that DNA relaxation in cells caused by the expression of LetD protein induces heat shock proteins via increased synthesis of ³².