Survival and development of immature stages of the Mediterranean fruit fly (Diptera: Tephritidae) in citrus fruit

We studied, under laboratory conditions, the performance of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), immature stages in intact whole fruit of three sweet orange varieties, lemon, and bitter oranges. Both citrus variety and fruit part (flavedo, albedo, and pulp) had strong effects on larval performance, smaller effects on pupae, and no effects on eggs. Fruit peel was the most critical parameter for larval development and survival, drastically affecting larval survival (inducing very high mortality rates). Among fruit regions, survival of larvae placed in flavedo was zero for all varieties tested except for bitter orange (22.5% survival), whereas survival in albedo was very low (9.8-17.4%) for all varieties except for bitter orange (76%). Survival of pupae obtained from larvae placed in the above-mentioned fruit regions was high for all varieties tested (81.1-90.7%). Fruit pulp of all citrus fruit tested was favorable for larval development. The highest survival was observed on bitter oranges, but the shortest developmental times and heaviest pupae were obtained from orange cultivars. Pulp chemical properties, such as soluble solid contents, acidity, and pH had rather small effects on larval and pupal survival and developmental time (except for juice pH on larvae developmental duration), but they had significant effects on pupal weight.     

Proteomic and metabolomic profiling of Valencia orange fruit after natural frost exposure

The aim of this study was to evaluate the response of orange fruit (Citrus sinensis var. Valencia Late) to freezing stress in planta, both immediately after the natural event and after a week, in order to understand the biochemical and molecular basis of the changes that later derive in internal and external damage symptoms. Using two‐dimensional differential gel electrophoresis to analyze exposed and non‐exposed fruit, 27 differential protein spots were detected in juice sacs and flavedo, among all comparisons made. Also, primary and secondary metabolites relative contents were analyzed in both tissues by gas chromatography‐mass spectrometry and liquid chromatography‐mass spectrometry, respectively. Proteins and compounds involved in regulatory functions, iron metabolism, oxidative damage and carbohydrate metabolism were the most affected. Interestingly, three glycolytic enzymes were induced by cold, and there was an increase in fermentation products (volatiles); all of that suggests that more energy generation might be required from glycolysis to counter the cold stress. Moreover, a notable increase in sugar levels was observed after frost, but it was not at the expense of organic acids utilization. Consequently, these results suggest a probable redistribution of photoassimilates in the frost‐exposed plants, tending to restore the homeostasis altered by that severe type of stress. Isosinensetin was the most cold‐sensitive secondary metabolite because it could not be detected at all after the frost, constituting a possible tool to early diagnose freezing damage.     

Novel expression patterns of carotenoid pathway-related genes in citrus leaves and maturing fruits

Carotenoids are abundant in citrus fruits and vary among cultivars and species. In the present study, high performance liquid chromatography (HPLC) and real-time polymerase chain reaction (PCR) were used to investigate the expression patterns of 23 carotenoid biosynthesis gene family members and their possible relation with carotenoid accumulation in fresh flavedo, juice sacs and leaves of Valencia orange during fruit maturation. Violaxanthin and lutein mainly accumulated in fruit (flavedo and juice sacs) and leaves (young and mature), respectively, accounting for nearly 79 %, 57 %, 53 % and 70 % of corresponding total carotenoids in February. Violaxanthin content quickly began to increase in flavedo in December, but the increase in juice sacs began later in January. In mature leaves, lutein content was three times that in young leaves; α-carotene and β-carotene were also much higher in mature leaves than in flavedo or juice sacs. Generally most of the carotenoid biosynthesis gene members were expressed at higher levels in flavedo than in juice sacs, and the expression of some continued to increase in flavedo during fruit maturation. All CHYB members expressed at high levels and had similar patterns in juice sacs. Interestingly, the capsanthin capsorubin synthase (CCS) members had similar expression levels and patterns in flavedo and juice sacs. Differences in gene expression between leaf and fruit tissues were noted, pointing to some tissue specificity for certain members of the gene families associated with carotenogenesis. The expression patterns of these 23 citrus carotenoid biosynthesis gene members were also compared with their expression patterns in other plants. Taken together, these first-hand expression data will be useful to define the tissue-specific roles of each gene member in accumulation of different carotenoids in citrus leaves and maturing fruits.     

Physiological changes associated with senescence and abscission in mature citrus fruit induced by 5-chloro-3-methyl-4-nitro-1H-pyrazole and ethephon application

This research compares effects of the compound 5-chloro-3-methyl-4-nitro-1 H -pyrazole (CMNP), a plant growth regulator that selectively promotes abscission in mature citrus fruit ( Citrus sinensis ), and the ethylene-releasing agent ethephon (2-chloroethylphosphonic acid). Application of CMNP and ethephon to mature citrus fruit reduced fruit detachment force and changed peel color from green to orange. More total chlorophyll was extracted from flavedo in early season (November) than late season (January), and both compounds caused a similar reduction in chlorophyll. In contrast, total carotenoid content was similar in November and January. Both abscission compounds increased total carotenoids, but induction was greater in January, and CMNP was more effective in both months. Phospholipase A₂ (PLA₂) activity increased after CMNP but not ethephon application. Electrolyte leakage increased 2 h after CMNP treatment, and total protein content was reduced by 50% after 72 h. Ethephon caused only minor changes in electrolyte leakage and total protein content. Inhibition of PLA₂ activity with aristolochic acid did not reduce leakage but inhibited total protein loss and reduced visual peel damage associated with CMNP. Ultrastructural observations indicated decreased number, and length of starch grains 3 h after CMNP treatment. A transient increase in soluble sugars was measured 3 h after CMNP application. Ethephon had little effect on soluble sugar content and changes in starch grains. Collectively, the results indicate that CMNP and ethephon induced color change in peel and advanced mature fruit abscission. However, CMNP but not ethephon promoted other physiological changes associated with senescence.     

Characterization of chitinases and β-1,3-glucanases in grapefruit flavedo during fruit development

Chitinase (EC 3.2.1.14) and β-1,3-glucanase (EC 3.2.1.39) activities in the flavedo of grapefruit ( Citrus paradisi cv. Marsh) were determined at 17 times during the course of fruit development. Chitinase activity is initially high in flavedo, but drops rapidly and is low, although fairly constant throughout the remainder of fruit development. In contrast to chitinase, β-1,3-glucanase activity is lowest in young fruit and increases during development. Western blots of crude flavedo extracts following SDS-PAGE were probed with antibodies raised against purified citrus chitinase and β-1,3-glucanase. Results of immunostaining revealed that changes in the activities of chitinase and β-1,3-glucanase were reflected in the amount of chitinase and glucanase protein present in the extracts. Only a single chitinase band was detected on western blots of crude flavedo extracts, whereas one glucanase band was present in young fruit and a second one appeared later in older fruit. Partial purification of flavedo chitinases and glucanases was performed using extracts prepared from immature and mature fruit for the two enzymes, respectively. Acidic and basic forms of both enzymes were present in the extracts; acidic and basic forms of chitinase were present in nearly equal amounts whereas basic glucanases predominated (91% of total activity). Acidic and basic chitinases differed in substrate specificity as well as products of degradation indicating the heterogeneous nature of the enzymes. Both acidic and basic glucanases required the presence of β-1,3 linkages for activity, were active against both soluble and insoluble β-1,3 glucans and generated similar products.     

Are citrus species favorable hosts for the Mediterranean fruit fly? A demographic perspective

We studied, under laboratory conditions, demographic parameters of adult Mediterranean fruit flies, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (medfly), obtained from three sweet orange varieties, lemon, and bitter oranges. These data were combined with immature developmental rates and survival on the same hosts to estimate host‐specific population parameters. Pairs of newly emerged adults from each citrus variety were held individually in transparent plastic cages, and females were allowed to oviposit in either red domes (artificial, pre‐punctured plastic oviposition devices), or intact, whole citrus fruits. We found strong effects of larval host (citrus fruits) on adult longevity and fecundity. In all five citrus varieties, females did not manage to deposit eggs into fruit pulp. The proportion of eggs laid in either the flavedo or albedo area of the fruit peel differed depending on the citrus variety. In all cases except bitter oranges, females oviposited fewer eggs in citrus fruits than in the artificial oviposition substrates, suggesting that most citrus fruits cause a significant reduction in the reproductive potential of medflies. Negative correlations were found between fecundity and (a) the density of oil glands, and (b) the amount of essential oils in the flavedo area of citrus fruits. There was no correlation between fecundity and other fruit physical characteristics, such as resistance of fruit peel to pressure and thickness of the flavedo. Apparently, resistance of citrus fruits to medfly infestation is directly related to citrus essential oils. The intrinsic rate of increase (r) was higher in bitter oranges than in the three sweet orange varieties tested. A negative r was estimated for flies that developed and oviposited in lemons, indicating a tendency for population decrease in this host. The suitability of citrus fruits for medfly development and the practical implications of our findings for management of medflies in citrus orchards are discussed.     

Essential oils of citrus fruit stimulate oviposition in the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae)

Female Mediterranean fruit flies (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) perceive both qualitative and quantitative aspects of citrus fruit chemistry. However, the behavioural and biological adjustments of this response remain largely unknown. In the present study, the ovipositional responses of gravid female medflies to essential oils (i.e. the most critical resistance factor to medfly infestation) of various citrus fruits are investigated. In dual‐choice (treatment versus distilled water control) experiments, females lay significantly more eggs into pre‐punctured hollow oviposition hemispheres (domes) provided with 1 µL of citrus peel oil from sweet orange, satsuma mandarin, bitter orange, grapefruit and lemon compared with odourless domes. No‐choice tests show a weak effect of lemon essential oils in stimulating oviposition. The female ovipositional response to sweet orange oil (the most active in eliciting oviposition) is dose‐dependent. Additionally, limonene, the most abundant chemical in all citrus oils, stimulates oviposition, whereas linalool, a representative compound of immature citrus fruit associated with high toxicity against immature stages of fruit flies, has a significant deterrent effect. In further no‐choice tests, females lay approximately 23% fewer eggs in limonene (93%) (amount found in orange oil) and 60% fewer eggs in limonene 93% plus linalool 3% (approximately 10‐fold the amount found in orange oil) mixtures, relative to sweet orange oil. The results suggest that the limonene content accounts largely (but not completely) for the ovipositional responses observed in sweet orange oil, whereas high linalool proportions are capable of significantly masking and/or disrupting its stimulatory effects in citrus oils. The importance and practical implications of these findings with respect to understanding how citrus fruit chemistry influences the ovipositional responses of medfly is discussed.     

Citrus abscission and Arabidopsis plant decline in response to 5-chloro-3-methyl-4-nitro-1H-pyrazole are mediated by lipid signalling

The compound 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP) is a pyrazole-derivative that induces abscission selectively in mature citrus (Citrus sinensis) fruit when applied to the canopy and has herbicidal activity on plants when applied to roots. Despite the favourable efficacy of this compound, the mode of action remains unknown. To gain information about the mode of action of CMNP, the effect of application to mature citrus fruit and Arabidopsis thaliana roots was explored. Peel contact was essential for mature fruit abscission in citrus, whereas root drenching was essential for symptom development and plant decline in Arabidopsis. CMNP was identified as an uncoupler in isolated soybean (Glycine max) mitochondria and pea (Pisum sativum) chloroplasts and an inhibitor of alcohol dehydrogenase in citrus peel, but not an inhibitor of protoporphyrinogen IX oxidase. CMNP treatment reduced ATP content in citrus peel and Arabidopsis leaves. Phospholipase A₂ (PLA₂) and lipoxygenase (LOX) activities, and lipid hydroperoxide (LPO) levels increased in flavedo of citrus fruit peel and leaves of Arabidopsis plants treated with CMNP. An inhibitor of PLA₂ activity, aristolochic acid (AT), reduced CMNP-induced increases in PLA₂ and LOX activities and LPO levels in citrus flavedo and Arabidopsis leaves and greatly reduced abscission in citrus and delayed symptoms of plant decline in Arabidopsis. However, AT treatment failed to halt the reduction in ATP content. Reduction in ATP content preceded the increase in PLA₂ and LOX activities, LPO content and the biological response. The results indicate a link between lipid signalling, abscission in citrus and herbicidal damage in Arabidopsis.     

Characterization of Pinalate,a novel Citrus sinensis mutant with a fruit-specific alteration that results in yellow pigmentation and decreased ABA content

The characterization of a novel mutant, named Pinalate, derived from the orange (Citrus sinensis L. Osbeck) Navelate, which produces distinctive yellow fruits instead of the typical bright orange colouration, is reported. The carotenoid content and composition, and ABA content in leaf and flavedo tissue (coloured part of the skin) of fruits at different developmental and maturation stages were analysed. No important differences in leaf carotenoid pattern of both phenotypes were found. However, an unusual accumulation of linear carotenes (phytoene, phytofluene and zeta- carotene) was detected in the flavedo of Pinalate. As fruit maturation progressed, the flavedo of mutant fruit accumulated high amounts of these carotenes and the proportion of cyclic and oxygenated carotenoids was substantially lower than in the parental line. Full-coloured fruit of Pinalate contained about 44% phytoene, 21% phytofluene, 25% zeta-carotene, and 10% of xanthophylls, whereas, in Navelate, 98% of total carotenoids were xanthophylls and apocarotenoids. The ABA content in the flavedo of Pinalate mature fruit was 3-6 times lower than in the corresponding tissue of Navelate, while no differences were found in leaves. Other maturation processes were not affected in Pinalate fruit. Taken together, the results indicate that Pinalate is a fruit-specific alteration defective in zeta-carotene desaturase or in zeta-carotene desaturase-associated factors. Possible mechanisms responsible for the Pinalate phenotype are discussed. Because of the abnormal fruit-specific carotenoid complement and ABA deficiency, Pinalate may constitute an excellent system for the study of carotenogenesis in Citrus and the involvement of ABA in fruit maturation and stress responses.     

Metabolism of indole-3-acetic acid by orange (Citrus sinensis) flavedo tissue during fruit development

[5-3H, 1'-14C, 13C6, 12C] Indole-3-acetic acid (IAA), was applied to the flavedo (epicarp) of intact orange fruits at different stages of development. After incubation in the dark, at 25 degrees C, the tissue was extracted with MeOH and the partially purified extracts were analyzed by reversed phase HPLC-RC. Six major metabolite peaks were detected and subsequently analyzed by combined HPLC-frit-FAB MS. The metabolite peak 6 contained oxindole-3-acetic acid (OxIAA), indole-3-acetyl-N-aspartic acid (IAAsp) and also indole-3-acetyl-N-glutamic acid (IAGlu). The nature of metabolite 5 remains unknown. Metabolites 3 and 4 were diastereomers of oxindole-3-acetyl-N-aspartic acid (OxIAAsp). Metabolite 2 was identified as dioxindole-3-acetic acid and metabolite 1 as a DiOx-IAA linked in position three to a hexose, which is suggested to be 3-(-O-beta-glucosyl) dioxindole-3-acetic acid (DiOxIAGlc). Identification work as well as feeding experiments with the [5-3H]IAA labeled metabolites suggest that IAA is metabolized in flavedo tissue mainly through two pathways, namely IAA-OxIAA-DiOxIAA-DiOxIAGlc and IAA-IAAsp-OxIAAsp. The flavedo of citrus fruit has a high capacity for IAA catabolism until the beginning of fruit senescence, with the major route having DiOxIAGlc as end product. This capacity is operative even at high IAA concentrations and is accelerated by pretreatment with the synthetic auxins 2,4-D, NAA and the gibberellin GA3.     

Comparative response to citrus foliage and citrus fruit odour by wild and mass-reared sterile Mediterranean fruit fly males of a genetic sexing strain

In greenhouse behavioural assessments conducted at Seibersdorf, Austria that allowed flies the choice of different types of potted host trees and plants, it was found that mature laboratory mass-reared Mediterranean fruit fly males, Ceratitis capitata (Wied), (Dipt. Tephritidae), strain Vienna-42 (temperature-sensitive lethal genetic sexing strain) showed the same preference for orange foliage, over other plants types and parts, as wild males. They aggregated in leks, released pheromone, and courted approaching females almost exclusively on orange foliage. Even though a number of other trees and plants were available to Vienna-42 males, less than 1% of these sexual activities occurred on the foliage or other plant parts of non-Citrus trees and not a single mating occurred there. In choice tests carried out in Chios, Greece in large field cages housing naturally planted orange trees, it was determined that mature and sterile Mediterranean fruit fly males of the same sexing strain responded to the odour emanating from the flavedo (due to superficial cuts made in the fruit peel containing the oil glands) of ripening sweet, sour oranges and lemons, in a similar intensity, order of fruit preference and time of day as did mature males of the local wild population. Males of both strains were frequently observed contacting with their mouth parts the oils exuding from the cuts made in the flavedo of the orange peel, or exhibiting homosexual activity on, or near the cuts. The results show that mass-reared, sterile males of the Vienna-42, genetic sexing strain behave in a comparable way to wild males in an important component of habitat location (odour of host foliage and host fruit) and hence are competitive with them in these respects. None of the standard international quality control tests for sterile Mediterranean fruit flies evaluates this important habitat location behaviour, which is a prerequisite for the successful encounter of the sexes in a sterile release programme.     

Chlorophyllase activity in chlorophyll-free citrus chromoplasts

Chromoplast fractions from mature, chlorophyll-less ‘Valencia’ orange (Citrus sinensis L. Osbeck) flavedo (= the outer coloured layer of citrus peel) showed considerable chlorophyllase activity. Acetone powders prepared from chromoplast fractions had 2.5× higher specific activity than those prepared from whole flavedo. Exposure of mature, chlorophyll-less fruit to ethylene caused a 2.5 to 4.0 fold increase in chlorophyllase activity. Juice chromoplasts showed negligible chlorophyllase activity. The results suggest that chlorophyllase activity as well as its induction by ethylene are not dependent upon the presence of chlorophyll in the tissue.     

Citrus chlorophyllase dynamics at ethylene-induced fruit color-break: a study of chlorophyllase expression, posttranslational processing kinetics, and in situ intracellular localization

Fruit color-break is the visual manifestation of the developmentally regulated transition of chloroplasts to chromoplasts during fruit ripening and often involves biosynthesis of copious amounts of carotenoids concomitant with massive breakdown of chlorophyll. Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. Here, we present the dynamics of native chlorophyllase (Chlase) and chlorophyll breakdown in lemon (Citrus limon) fruit during ethylene-induced color-break. We show, using in situ immunofluorescence on ethylene-treated fruit peel (flavedo) tissue, that citrus Chlase is located in the plastid, in contrast to recent reports suggesting cytoplasmic localization of Arabidopsis (Arabidopsis thaliana) Chlases. At the intra-organellar level, Chlase signal was found to overlap mostly with chlorophyll fluorescence, suggesting association of most of the Chlase protein with the photosynthetic membranes. Confocal microscopy analysis showed that the kinetics of chlorophyll breakdown was not uniform in the flavedo cells. Chlorophyll quantity at the cellular level was negatively correlated with plastid Chlase accumulation; plastids with reduced chlorophyll content were found by in situ immunofluorescence to contain significant levels of Chlase, while plastids containing still-intact chlorophyll lacked any Chlase signal. Immunoblot and protein-mass spectrometry analyses were used to demonstrate that citrus Chlase initially accumulates as an approximately 35-kD precursor, which is subsequently N-terminally processed to approximately 33-kD mature forms by cleavage at either of three consecutive amino acid positions. Chlase plastid localization, expression kinetics, and the negative correlation with chlorophyll levels support the central role of the enzyme in chlorophyll breakdown during citrus fruit color-break.     

Phytohormone Changes and Carbohydrate Status in Sweet Orange Fruit from Huanglongbing-infected Trees

Huanglongbing (HLB) infection alters citrus fruit growth and development, resulting in small, misshapen, and poorly colored fruit containing aborted or partially developed seeds. Typically, symptomatic fruit have delayed maturation and abscise prematurely. We studied carbohydrate and phytohormone changes in HLB-affected fruit to explain symptom development because (1) carbohydrate shortage has been linked to fruit growth arrest and eventually abscission and (2) hormonal signals regulate, at least partially, fruit set and development. Symptomatic fruit (S), asymptomatic fruit (AS) from symptomatic trees, and healthy fruit (H) from asymptomatic trees were harvested from ‘Valencia’ sweet orange trees [Citrus sinensis (L.) Osbeck] infected with the HLB pathogen or not, as verified by PCR. Mature S weighed less, had lower °Brix, were smaller, had more aborted seeds, and were greener than AS or H. Starch and sucrose contents were lower in mature S flavedo compared with that of H and AS. S and AS harvested 7 and 12 months after full bloom produced significantly less ethylene than H. Indole-3-acetic acid (IAA) and abscisic acid (ABA) contents in flavedo removed from the stylar end, middle section, or stem end of fruit generally were higher in S flavedo than in AS and H. ABA content was fourfold higher in flavedo from the middle section of S than in AS and H. Flavedo excised from the large shoulder of misshapen S had significantly higher IAA content when compared with the normal-sized area of the same fruit on the opposite side. This increase corresponded to an increase in hypodermal cell area in S flavedo. Overall, these data reveal an imbalance of carbohydrate and phytohormone status in fruit from HLB-infected trees and suggest a role of such changes in fruit symptom development.     

Insecticidal activity of Citrus aurantium fruit, leaf, and shoot extracts against adult olive fruit flies (Diptera: Tephritidae)

Solvent extracts of differing polarity from Citrus aurantium (L.) (Rutaceae) fruit, leaves, and shoots were evaluated for biological activity against adults of the olive fruit fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae). Using a petri dish residual exposure bioassay, we found that the petroleum ether extract from fruit alone showed insecticidal activity against the flies. The extract of the three fruit tissues (flavedo [peel], albedo, and flesh) indicated that bioactivity was limited to the flavedo, and this activity was significantly higher than that of the whole fruit extract. The most effective extract was obtained when fresh flavedo was used, whereas extracts of oven-dried flavedo were inactive. Fruit maturity also affected bioactivity; extracts of ripe fruit were more effective than those of unripe fruit. Our results suggest that C. aurantium flavedo contains secondary metabolites with insecticidal activity against B. oleae adults.     

Flavonoid composition of fruit tissues of citrus species

An HPLC analysis was performed on the concentrations of flavonoids in 42 species and cultivars of the Citrus genus and those of two Fortunella and one Poncirus species according to the classification system established by Tanaka. The composition of 8 flavanones and 9 flavone/ols for these species was determined in the albedo, flavedo, segment epidermis and juice vesicle tissues, and those in the fruit and peel tissues were calculated from the composition data of the tissues. A principal component analysis showed that such neohesperidosyl flavonoids as neoeriocitrin, naringin, neohesperidin, and rhoifolin had large factor loading values in the first principal component for each tissue. The flavonoid composition of citrus fruits was approximately the same within each section of Tanaka's system, except for the species in the Aurantium section and those with a peculiar flavonoid composition such as Bergamot (C. bergamia), Marsh grapefruit (C. paradisi), Sour orange (C. aurantium), and Shunkokan (C. shunkokan). The Aurantium section included both naringin-rich and hesperidin-rich species.     

Response of Ceratitis capitata to citrus chemicals under semi-natural conditions

Responses of Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) adults to chemicals from citrus fruits were quantified in choice tests conducted in large field cages housing naturally planted orange trees, on the Greek island of Chios. Both sexes responded strongly to chemicals released from artificial cuts made in the pulp of peeled oranges and also to natural or commercial orange juice applied to the surface of yellow 7.0 cm diameter spheres. Males also responded strongly to chemicals released from cuts made in the oily region of the orange peel, or flavedo and less to sour orange and lemon. In contrast, females did not respond to flavedo chemicals. Male response to peel chemicals occurred throughout the day and increased as the number of cuts per fruit increased from 2 to 8. Contact of male mouthparts with sap from cuts, and mating attempts between males on or near the cuts were frequently observed. The males also responded to commercial essential orange oils applied to yellow spheres, but less to essential mandarin oils. The role of citrus chemicals in host finding behaviour of C. capitata and their potential use for the development of effective traps for monitoring and controlling the fly are discussed. The experimental method followed in this study can be used as a standard technique for evaluation of the quality of flies to be used in sterile insect technique programmes.