Unlocking the full power of electrochemical fingerprinting for on-site sensing applications.

Electrochemical sensing for the semi-quantitative detection of biomarkers, drugs, environmental contaminants, food additives, etc. shows promising results in point-of-care diagnostics and on-site monitoring. More specifically, electrochemical fingerprint (EF)-based sensing strategies are considered an inviting approach for the on-site detection of low molecular weight molecules. The fast growth of electrochemical sensors requires defining the concept of direct electrochemical fingerprinting in sensing. The EF can be defined as the unique electrochemical signal or pattern, mostly recorded by voltammetric techniques, specific for a certain molecule that can be used for its quantitative or semi-quantitative identification in a given analytical context with specified circumstances. The performance of EF-based sensors can be enhanced by considering multiple features of the signal (i.e., oxidation or reduction patterns), in combination with statistical data analysis or sample pretreatments or by including electrode surface modifiers to enrich the EF. In this manuscript, some examples of EF-based sensors, strategies to improve their performances, and open challenges are discussed to unlock the full power of electrochemical fingerprinting for on-site sensing applications. Graphical abstract Electrochemical fingerprint-based sensing strategies can be used for the detection of electroactive analytes, such as antibiotics, phenolic compounds, and drugs of abuse. These strategies show selective and sensitive responses and are easily combined with portable devices.

Heated-controlled atmosphere postharvest treatments for Macchiademus diplopterus (Hemiptera: Lygaeidae) and Phlyctinus callosus (Coleoptera: Curculionidae).

Nonchemical, environmentally friendly quarantine treatments are preferred for use in postharvest control of insect pests. Combined high temperature and controlled atmosphere quarantine treatments for phytosanitary fruit pests Macchiademus diplopterus (Distant) (Hemiptera: Lygaeidae) and Phlyctinus callosus (Schoenherr) (Coleoptera: Curculionidae) were investigated to determine the potential of such treatments for quarantine security. Field-collected, aestivating M. diplopterus adults and P. callosus adults were treated using a controlled atmosphere waterbath system. This system simulates the controlled atmosphere temperature treatment system (CATTS) used to control a number of phytosanitary pests in the United States and allows for a rapid assessment of pest response to treatment. Insects were treated under regular air conditions and a controlled atmosphere of 1% oxygen, 15% carbon dioxide in nitrogen, at two ramping heat rates, 12 and 24 degrees C/h. Treatment of both species was more effective under both heating rates when the controlled atmosphere condition was applied. Under these conditions of controlled atmospheres, mortality of P. callosus was greater when the faster heating rate was used, but the opposite was true for M. diplopterus. This could be due to the physiological condition of aestivation contributing to metabolic arrest in response to the stresses being applied during treatment. Results indicate that the potential for the development of CATTS treatments for these phytosanitary pests, particularly P. callosus, is promising.

Potential of heated controlled atmosphere postharvest treatments for the control of Thaumatotibia leucotreta (Lepidoptera: Tortricidae).

Controlled atmosphere/temperature treatment system (CATTS) is an environmentally friendly postharvest mitigation treatment that uses high temperature forced-air combined with a low oxygen and high carbon dioxide atmosphere to control quarantine pests. The development of CATTS treatments is expensive and time-consuming. For a more rapid assessment of different species and life stages' tolerances to heated controlled atmospheres, the controlled atmosphere water bath (CAWB) system can be used to help advance the development of CATTS treatments for pests. The CAWB system was used to test the response of eggs and larval stages of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae). Eggs and larvae at different developmental stages were treated under regular air and a modified controlled atmosphere of 1% O2 and 15% CO2, at two ramping heat rates: 12 and 24 degrees C/h. Typically the faster heat rate and modified atmosphere reduced treatment times required to control the different life stages. T. leucotreta larvae were more tolerant of the treatments than eggs. The most tolerant life stage was the fourth instar. Effective treatments against the most tolerant life stage determined by the CAWB system can now be used to develop CATTS technology against T. leucotreta. Further research will focus on developing CATS treatments using infested fruit to determine effective treatments that maintain fruit quality.

Efficacy of the biofumigant fungus Muscodor albus (Ascomycota: Xylariales) for control of codling moth (Lepidoptera: Tortricidae) in simulated storage conditions.

Codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), a serious pest of pome fruit, is a threat to exportation of apples (Malus spp.) because of the possibility of shipping infested fruit. The need for alternatives to fumigants such as methyl bromide for quarantine security of exported fruit has encouraged the development of effective fumigants with reduced side effects. The endophytic fungus Muscodor albus Worapong, Strobel and Hess (Ascomycota: Xylariales) produces volatile compounds that are biocidal for several pest organisms, including plant pathogens and insect pests. The objectives of our research were to determine the effects of M. albus volatile organic compounds (VOCs) on codling moth adults, neonate larvae, larvae in infested apples, and diapausing cocooned larvae in simulated storage conditions. Fumigation of adult codling moth with VOCs produced by M. albus for 3 d and incubating in fresh air for 24 h at 25 degrees C resulted in 81% corrected mortality. Four- and 5-d exposures resulted in higher mortality (84 and 100%, respectively), but control mortality was also high due to the short life span of the moths. Exposure of neonate larvae to VOCs for 3 d on apples and incubating for 7 d resulted in 86% corrected mortality. Treated larvae were predominantly first instars, whereas 85% of control larvae developed to second and third instars. Exposure of apples that had been infested for 5 d, fumigated with M. albus VOCs for 3 d, and incubated as described above resulted in 71% corrected larval mortality. Exposure of diapausing cocooned codling moth larvae to VOCs for 7 or 14 d resulted in 31 and 100% mortality, respectively, with negligible control mortality. Our data on treatment of several stages of codling moth with M. albus VOCs indicate that the fungus could provide an alternative to broad spectrum chemical fumigants for codling moth control in storage and contribute to the systems approach to achieve quarantine security of exported apples.

DNA diagnostics to identify internal feeders (Lepidoptera: Tortricidae) of pome fruits of quarantine importance.

A diagnostic polymerase chain reaction (PCR) method is presented for differentiating among the North American internal apple-feeding pests codling moth, Cydia pomonella (L.); oriental fruit moth, Grapholita molesta (Busck); lesser appleworm, Grapholita prunivora (Walsh); and cherry fruitworm, Grapholita packardi Zeller. An approximately 470-bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in three to six specimens of each species. Consistent and diagnostic differences were observed among the species in two regions of COI from which forward and reverse primers were designed to amplify a 112-116-bp segment of the gene. The primer sets were used to selectively amplify DNA from specimens of diverse geographic origin for each corresponding target species. Protocols were adapted for conventional and quantitative PCR, the latter being substantially faster. The method was validated as a decision-making tool for quarantine identifications for Mexico by representatives of their phytosanitary agency (Sanidad Vegetal). The method can facilitate identification of intercepted internal feeding Lepidoptera in apple and pear for many other importing nations.

Active regulation of respiration and circulation in pupae of the codling moth (Cydia pomonella).

Regulation of autonomic physiological functions has been investigated by means of multisensor electronic methods, including electrocardiographic recording of heartbeat, strain-gauge recording of extracardiac hemocoelic pulsations (EHPs), anemometric recording of air passage through spiracles and respirographic recording of O(2) consumption and CO(2) output. Pupae of Cydia exhibit continuous respiration without remarkable bursts of CO(2). The dorsal vessel of these pupae exhibited regular heartbeat reversals characterized by shorter intervals of faster (forward oriented or anterograde) pulsations and longer intervals of slower (backward oriented or retrograde) peristaltic waves. The periodically repeated EHPs were present during the whole pupal interecdysial period. The internal physiological mechanisms regulating the cardiac (heartbeat) and extracardiac (EHP) pulsations were completely independent for most of the pupal instar. Simultaneous multisensor analysis revealed that the anterograde heartbeat of the dorsal vessel had similar but not identical frequency with EHPs. During advanced pharate adult development, frequency of cardiac and extracardiac pulsation periods profoundly increased until almost uninterrupted pulsation activity towards adult eclosion. At this time, the cardiac and extracardiac pulsations occasionally performed in concert, which enhanced considerably the efficacy of hemolymph circulation in pharate adults with high metabolic rates. The fastest hemolymph flow through the main body cavity was always associated with EHPs and with anterograde heartbeat. Simple physical diffusion of O(2) and CO(2) through spiracles (diffusion theory of insect respiration) does not play a significant role in pupal respiration. Instead, several kinds of regulated, mechanical ventilations of the tracheal system, including EHPs are responsible for effective tracheal ventilation.

Laboratory rearing of lesser appleworm (Lepidoptera: Tortricidae).

The lesser appleworm, Cydia prunivora (Walsh), was reared successfully in the laboratory. Larvae of various instars were collected in the field from hawthorn fruit, Crataegus spp. Initially, immature apples served as the food source for the larvae in the laboratory. Rearing was conducted in a greenhouse and later in combination with a controlled environment room at 25 degrees C, 50-60% RH, and a photoperiod of 18:6 (L:D) h. Under these conditions, a generation required approximately 30 d. Fifty-six adult lesser appleworm moths emerged from the original field collected hawthorn fruits. After a decline in the number of the F1 generation to 39 moths, the colony on mature apples, increased to in excess of 10,000 moths by the fifth generation with a mean survival rate to adult of 68.0%. When production on immature apples was compared with that on four artificial diets, the most promising of the artificial diets was the lima bean-based diet currently used to rear the oriental fruit moth, Cydia molesta (Busck), with a mean survival rate of 46.4%. The other bean-based diets tested were not as satisfactory. Pear foliage was the preferred oviposition substrate of those tested, including apple and hawthorn foliage. No eggs were deposited on plain waxed paper or glass microscope slides; however, large numbers of eggs were deposited on waxed paper treated with a water extract of pear foliage and immature apples.

Sub-zero cooling synchronizes post-diapause development of codling moth, Cydia pomonella.

Sub-zero cooling treatments of -10 degree C and -15 degree C for 2-6 days were evaluated as a means of meeting the chilling requirement of diapause and to synchronize post-diapause development in larvae which were held in diapause for less than 6 months. Sub-zero cooling did not affect male longevity. After six months in diapause, diapause/cooled females were longer lived than diapause control females but not longer than non-diapause females. In general, diapause-cooled males passed more spermatophores than males from control diapause and non-diapause groups. In general, sub-zero cooling did not consistently affect fecundity and % egg hatch. Non-diapause females laid the most eggs after six months in diapause, and diapause-cooled females laid the most eggs after seven months in diapause. The duration of sub-zero cooling had a significant effect on post-diapause emergence in relation to the duration that the larvae were in diapause. Sub-zero cooling for 4 days at -10 degree C significantly reduced the number of days to adult emergence of larvae which had been in diapause for 0-4 months. Sub-zero cooling at -15 degree C for durations of 2, 4, and 6 days had more variable effects on emergence, but in most cases, sub-zero cooling reduced the amount of time to and span of adult emergence.

Characterization of a gene for spinach CAP160 and expression of two spinach cold-acclimation proteins in tobacco.

The cDNA sequence for CAP160, an acidic protein previously linked with cold acclimation in spinach (Spinacia oleracea L.), was characterized and found to encode a novel acidic protein of 780 amino acids having very limited homology to a pair of Arabidopsis thaliana stress-regulated proteins, rd29A and rd29B. The lack of similarity in the structural organization of the spinach and Arabidopsis genes highlights the absence of a high degree of conservation of this cold-stress gene across taxonomic boundaries. The protein has several unique motifs that may relate to its function during cold stress. Expression of the CAP160 mRNA was increased by low-temperature exposure and water stress in a manner consistent with a probable function during stresses that involve dehydration. The coding sequences for CAP160 and CAP85, another spinach cold-stress protein, were introduced into tobacco (Nicotiana tabacum) under the control of the 35S promoter using Agrobacterium tumefaciens-based transformation. Tobacco plants expressing the proteins individually or coexpressing both proteins were evaluated for relative freezing-stress tolerance. The killing temperature for 50% of the cells of the transgenic plants was not different from that of the wild-type plants. As determined by a more sensitive time/temperature kinetic study, plants expressing the spinach proteins had slightly lower levels of electrolyte leakage than wild-type plants, indicative of a small reduction of freezing-stress injury. Clearly, the heterologous expression of two cold-stress proteins had no profound influence on stress tolerance, a result that is consistent with the quantitative nature of cold-stress-tolerance traits.

Effects of heating rate on the mortality of fifth-instar codling moth (Lepidoptera: Tortricidae).

Models were developed to describe the effects of heating rate during heat treatments on the mortality of 5th-instar codling moth, Cydia pomonella (L.). An old model, developed from previous studies over a limited range of heat treatments, was 1st formulated. Subsequent heat treatments, using a computerized water bath system and linear heating rates of 4, 6, 8, 10, and 12 degrees C/h at 42, 44, and 46 degrees C, were used to test the old model. The mortality data from the water bath study were used to develop a new model. Although the old model provided a good estimate of the effects of heating rate on 5th-instar mortality, it overestimated mortality at midrange heating rates. Also, the old model was awkward to use because it required a correction for each treatment temperature. The new model incorporated treatment temperature into the equation, and was more accurate and easier to use. It was determined that the slower the rate of heating, the longer the exposure to the final treatment temperature was needed to achieve 95% mortality.

Respiratory response of fifth-instar codling moth (Lepidoptera: Tortricidae) to rapidly changing temperatures.

Fifth-instar codling moth, Cydia pomonella (L.), larvae were exposed to 10 simulated heat treatments of apples and pears and CO2 levels were monitored as a measure of respiration. Marked increases in respiration rates (microliter CO2/mg/min) were noted during these treatments. Respiration peaked between 3.5 and 4.8 microliters CO2/mg/min; the amount of time to peak respiration depended on the heating rate and was correlated to the LT95. No differences were observed between male and female larvae in the timing of the peaks of CO2 production. In treatments where mortality occurred, CO2 levels dropped to zero, but only after a considerable time after death. Respiratory recovery rates, the time it took for CO2 levels to return to normal, were recorded after treatments at time points where CO2 production reached 3/4 and maximum peak. Respiration rates at constant temperatures were recorded within the range of 10-30 degrees C. Q10 over this range was 1.49, whereas Q10 was the greatest, 2.54, between 10 and 15 degrees C.

Characterization of a spinach gene responsive to low temperature and water stress.

The characterization of a cDNA for an 85 kDa spinach protein, CAP85 (cold acclimation protein) that is responsive to cold acclimation and water stress is described. Both transcript and protein levels are increased during cold acclimation and water stress. A novel characteristic of CAP85 is the presence of an 11 amino acid, lysine-rich repeat, common to Group 2 LEAs (late embryogenesis abundant proteins), which is included within a larger repeating motif present in 11 copies. Two other motifs of 8 and 16 residues are also found in three and four copies, respectively. CAP85 like other dehydrins and cold-regulated polypeptides remains soluble upon boiling. Protein blot analyses indicate that CAP85 protein is expressed in all aerial tissues as well as in roots. RNA blots show the presence of mRNA for the 85 kDa protein in leaf, petiole, and root tissue. Cell fractionation studies suggest that CAP85 is predominantly found in the cytosol.

Association of 70-kilodalton heat-shock cognate proteins with acclimation to cold.

Exposure of young spinach seedlings (Spinacia oleracea L. cv Bloomsdale) to 5 degrees C leads to an increase in the synthesis of several 79-kilodalton proteins that are present in leaf tissue grown at 20 degrees C. Protein sequence analyses and immunological cross-reactivity indicate that this group of proteins belongs to the 70-kilodalton heat-shock family. Steady-state transcript levels and protein synthesis are increased two- to threefold within 1 day, but immunoblot analyses suggest that the steady-state concentration of this protein group in leaf tissue only gradually accumulates at low temperature. It is proposed that the increased synthesis of several members of the 70-kilodalton heat-shock family could result from an influence of low temperature on protein folding and/or assembly processes.

Hydration-state-responsive proteins link cold and drought stress in spinach.

Spinach (Spinacia oleracea L.) seedlings exposed to low nonfreezing temperatures (0-10° C) that promote cold acclimation, synthesize a variety cold-acclimation proteins and at the same time acquire a greater ability to withstand cellular dehydration imposed by the freezing of tissue water. Two of these proteins (160 and 85 kDa) become more abundant over time at low temperature. In addition, a small decline in tissue water status from a maximally hydrated state also appears to be associated with an initiation of the accumulation of these proteins at a noninductive temperature. Imposing a severe water stress on young seedlings grown at 25° C by withholding water leads to substantial accumulation of the 160- and 85-kDa proteins, and maximal induction of freezing tolerance. This evidence implies that responses to cold acclimation and water stress involve common mechanisms, and further establishes the linkage of these two proteins with stresses having an osmotic component.

Familial hypercholesterolemia in a rhesus monkey pedigree: molecular basis of low density lipoprotein receptor deficiency.

We have recently identified a family of rhesus monkeys with members exhibiting a spontaneous hypercholesterolemia associated with a low density lipoprotein receptor (LDLR) deficiency. By using the polymerase chain reaction, we now show that the affected monkeys are heterozygous for a nonsense mutation in exon 6 of the LDLR gene. This mutation changes the sequence of the codon for amino acid 284 (tryptophan) from TGG to TAG, thereby generating a nonsense codon potentially resulting in a truncated 283-amino acid protein, which needs documentation, however. This G----A mutation also creates a site for the restriction endonuclease Spe I. Using this site as a marker for this nonsense mutation, we have shown that the mutation is present in all of the affected members of the pedigree and absent in unaffected members and that the mutation segregates with the phenotype of spontaneous hypercholesterolemia through three generations. Quantitative analyses of RNA obtained from liver biopsies show that the abundance of the LDLR RNA is also reduced by about 50%. Thus, we have identified a primate model for human familial hypercholesterolemia which will be useful for studying the relationship between the LDLR and lipoprotein metabolism and for assessing the efficacy of diets and drugs in the treatment of human familial hypercholesterolemia.

Rhesus monkey model of familial hypercholesterolemia: relation between plasma Lp[a] levels, apo[a] isoforms, and LDL-receptor function.

We previously described a family of rhesus monkeys in which three out of six members had a spontaneous hypercholesterolemia related to a decrease in number of low density lipoprotein receptors (LDL-R) (Scanu et al. 1988. J. Lipid Res. 29: 1671-1681). During the current work an additional female normocholesterolemic offspring was generated from the mating of the original dam and sire. Moreover, from the breeding of one of the affected male offspring with six unrelated normocholesterolemic female monkeys, eight offspring were generated of which three were hypercholesterolemic on a cholesterol-free diet and exhibited the same degree of LDL-R deficiency as shown by studies in skin fibroblast cultures. All of the animals studied had levels of plasma lipoprotein[a] protein ranging between 1.0 mg/dl and 57.5 mg/dl that were only weakly correlated with total plasma cholesterol, LDL cholesterol, and apoB. LDL-R deficiency correlated with plasma LDL but not Lp[a]. A 7 week fat challenge (16.5% lard, 0.64% cholesterol) that raised the plasma LDL levels markedly had no effect on plasma Lp[a]. Animals with the single band apo[a] phenotype moving on SDS-PAGE faster than apoB-100 exhibited a tendency for high plasma Lp[a] levels which, however, varied widely. Wide variations in Lp[a] levels were also noted with the other apo[a] phenotypes. Taken together our results demonstrate a successful transmission to second generation animals of the LDL-R deficiency phenotype and provide evidence that this phenotype correlates well with plasma LDL levels but not Lp[a]. Our data also suggest that the apo[a] gene is only partially involved in the regulation of the plasma Lp[a] levels.

Genetically determined hypercholesterolemia in a rhesus monkey family due to a deficiency of the LDL receptor.

A family of rhesus monkeys comprising a sire, a dam, and four male offspring were fed a cholesterol-free Purina Chow diet for several months. The sire, 431-J, and two of the offspring, B-8204 and B-8806, had persistent plasma cholesterol levels in the range of 100-130 mg/dl, whereas the dam, 766-I, and the two other offspring, B-1000 and B-7643, exhibited a marked hypercholesterolemia in the 250-300 mg/dl range associated with an elevation of plasma LDL and apoB. When fed for 12 weeks a diet containing 12.5% lard and 0.25% cholesterol, sire, dam, B-1000 and B-7643 exhibited a marked hypercholesterolemia (500-800 mg/dl range), whereas B-8204 and B-8806 developed only a modest hypercholesterolemia (200-250 mg/dl). All animals were Lp[a]+. Skin fibroblasts from each animal and from control cells were grown in 10% fetal calf serum, transferred to 10% lipoprotein-deficient serum for 48 hr, and then incubated at 4 degrees C or 37 degrees C with 125I-labeled Lp[a]-free LDL. The fibroblasts from dam and offspring B-1000 and B-7643 bound and internalized 125I-labeled LDL less efficiently than control cells. Mathematical analyses of the 4 degrees C binding data indicated that there were no significant differences in LDL binding affinity between test and control cells suggesting that cells from the animals with a spontaneous hypercholesterolemia had a decreased number of LDL receptors. This conclusion was supported by the results of ligand and immunoblot analyses carried out on cell lysates separated by gradient gel electrophoresis. We conclude that a genetically determined LDL receptor deficiency was responsible, in part, for the spontaneous hypercholesterolemia observed in three out of the six family members and that this deficiency accounted for the hyperresponsiveness to a dietary fat and cholesterol challenge by the dam and the two offspring, B-1000 and B-7643. The hyperresponsiveness noted in the sire that had no evidence for LDL-receptor deficiency illustrates that factors other than the LDL receptor were responsible for the hypercholesterolemia attending the fat challenge.