Probiotic based-diet effect on the immune response and induced stress in irradiated mass reared Ceratitis capitata males (Diptera: Tephritidae) destined for the release in the sterile insect technique programs.

Ceratitis capitata (medfly) is one of the most devastating crop pests worldwide. The Sterile Insect Technique (SIT) is a control method that is based on the mass rearing of males, their sterilization, and release in the field. However, the effectiveness of the technique depends on the quality of the released males and their fitness. We previously isolated and selected a probiotic bacteria (Enterobacter sp.), from wild-caught medflies, according to criteria that improved biological quality traits of reared medfly males.We firstly evaluated the impact of the irradiation on the expression of different immune and stress genes in the medfly sterile males. Expression was measured at differents time points ranging from 0 to 168 h after irradiation to capture the response of genes with distinct temporal expression patterns. Then, we supplemented the larval diet with previously isolated Enterobacter sp.strain, live and autoclaved at various concentrations to see whether the probiotic treatments affect, through their protective role, the gene expression level, and quality traits. The irradiation had significant effect on the genes attacin, cecropin, PGPR-LC, hsp23, and hsp70 level expression. The expression of attacin and PGPR-LC was up-regulated while that of cecropin was down-regulated. Hsp genes showed decreased levels between 0 and 18 h to peak at 72 h. However, the supplementation of the probiotic strain, either live or autoclaved, was statistically significant only for attacingene. However, significant interaction time x probiotic was noticed for attacin, cecropin, hsp23 and hsp70. The probiotic treatments also improved the quality control parameters like pupal weight. From this work we can conclude that a consortium of parabiotics (autoclaved probiotics) treatment will be recommended in insectaries considering both the beneficial effects on mass reared insects and its general safety for insectary workers and for environment.

Irradiation dose does not affect male reproductive organ size, sperm storage, and female remating propensity in Ceratitis capitata.

The Mediterranean fruit fly Ceratitis capitata is a globally invasive pest, often controlled with the sterile insect technique (SIT). For the SIT, mass-rearing of the target insect followed by irradiation are imperatives. Sterile males are often less able to inhibit female remating and transfer less number of sperm, and even irradiation could affect male reproductive organs, with consequences for their ability to inhibit female remating. On the other hand, male age could affect their ability to modulate female response after mating. Here, we evaluated the quality of the genetic sexing strain Vienna-8-tsl mass-reared in Bioplanta San Juan, Argentina, under laboratory conditions, with regard to: (i) the ability of sterile males irradiated at 100 or 140 Gy to inhibit female remating, in the same day and at 24 h of first copulation; (ii) the ability of 3, 4 or 5 day-old sterile males to inhibit female remating at 24 h of first copulation, and (iii) the effect of a reduction in irradiation doses on the number of sperm stored by females and reproductive organ size in virgin males. Sterile males were better able than wild males to inhibit female remating in the same day of first copulation and as able as wild males 1 day after first copulation. Male age did not affect their ability to inhibit female receptivity. Number of sperm stored by females, testes size and ectodermal accessory glands size were not affected by male identity, while sterile 100 Gy males had larger mesodermal accessory glands than control lab males. A reduction in irradiation dose does not impact any variable measured, except for percentage of sperm-depleted females: females mated with sterile 100 Gy males had lower probabilities to store sperm. The results showed here are very encouraging for tsl Vienna 8 strain reared in Argentina and are discussed in comparison with previous studies in C. capitata female remating with dissimilar results.

Light Conditions After Emergence Affect Food Consumption and Survival of Ceratitis capitata (Diptera: Tephritidae) Sterile Males.

To favor and standardize fruit fly production, many environmental factors are regulated in a mass-rearing facility. Specifically, in the holding rooms where sterile fruit flies are kept before releasing, they are exposed to constant darkness in order to reduce aggressive interactions and depletion of energy resources. However, such light conditions could negatively affect the quality traits and male mating performance. The objective of this study was to determine whether light conditions have an effect on quality traits in mass-reared 'Mediterranean fruit flies' Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Under laboratory conditions and in two sequential experiments, we studied the effect of 1) different light intensities and 2) photoperiods, on adult food consumption, adult body weight, survival, and percent of flying and calling males. In the first experiment, sterile male flies were exposed to different light intensities during 3 d after emergence: 1000, 500, or 250 lux, with a 12:12 photoperiod. Complete darkness was used as a control. In the second experiment, adult flies were exposed to the following photoperiods (L(1000 lux)-D): 14-10, 12-12, 8-16, 4-20, and 0-24 as a control. Our results showed that flies under darkness ingested less food, were heavier, and exhibited higher survival than flies under any treatment of light intensity or photoperiod. Furthermore, the percentage of fliers and number of calling males did not differ among treatments. We conclude that holding males for 3 d under darkness do not affect their quality; indeed, emerging under this condition appears to favor them. The implications of these findings for SIT programs are discussed.

Effect of Irradiation Doses on Sterility and Biological Security in a Genetically Modified Strain of the Mediterranean Fruit Fly (Diptera: Tephritidae).

The genetically modified strain of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) VIENNA 8 1260, was developed from the genetic sexing strain VIENNA 8. It has two molecular markers that exhibit red fluorescence in the body and green fluorescence in testis and sperm. These traits offer a precise tool to discriminate between mass-reared and wild males, increasing the effectiveness of sterile insect technique. The reproductive performance of the VIENNA 8 1260 and VIENNA 8 D53- (without the D53 inversion introduced to prevent recombination) was compared at different irradiation doses. The general effect of irradiation on VIENNA 8 1260 followed the same patterns documented in previous publications for VIENNA 8 D53-. Irradiation doses of 80 Gray or greater reduced fertility and induced high levels of sterility in wild females. Fecundity reduction was higher in VIENNA 8 1260 than in VIENNA 8 D53- females. Vertical transmission of the fluorescence gene was confirmed up to the F4 generation. Substerilization in the VIENNA 8 1260 could jeopardize the usefulness of the transgenic strain due to the possible vertical transfer of the fluorescence transgene from the sterile males to the wild flies. A biologically safe higher irradiation dose could result in reduced competitiveness of the VIENNA 8 1260 strain. Mating and remating experiments suggest that Mediterranean fruit fly females exhibit a relative precedence in the use of the sperm: though both sperms are mixed, sperm from the remating is spent first. Results suggest a lower fitness of VIENNA 8 1260 sperm, when compared with sperm from a nonfluorescent bisexual strain, which is consistent with the lower reproductive performance documented for the VIENNA 8 1260 strain.

A genomic perspective to assessing quality of mass-reared SIT flies used in Mediterranean fruit fly (Ceratitis capitata) eradication in California.

BACKGROUND: Temperature sensitive lethal (tsl) mutants of the tephritid C. capitata are used extensively in control programs involving sterile insect technique in California. These flies are artificially reared and treated with ionizing radiation to render males sterile for further release en masse into the field to compete with wild males and disrupt establishment of invasive populations. Recent research suggests establishment of C. capitata in California, despite the fact that over 250 million sterile flies are released weekly as part of the state's preventative program. In this project, genome-level quality assessment was performed, measured as expression differences between the Vienna-7 tsl mutants used in SIT programs and wild flies. RNA-seq was performed to provide a genome-wide map of the messenger RNA populations in C. capitata, and to investigate significant expression changes in Vienna-7 mass reared flies. RESULTS: Flies from the Vienna-7 colony showed a markedly reduced abundance of transcripts related to visual and chemical responses, including light stimuli, neural development and signaling pathways when compared to wild flies. In addition, genes associated with muscle development and locomotion were shown to be reduced. This suggests that the Vienna-7 line may be less competitive in mating and host plant finding where these stimuli are utilized. Irradiated flies showed several transcripts representing stress associated with irradiation. CONCLUSIONS: There are significant changes at the transcriptome level that likely alter the competitiveness of mass reared flies and provide justification for pursuing methods for strain improvement, increasing competitiveness of mass-reared flies, or exploring alternative SIT approaches to increase the efficiency of eradication programs.

Influence of modified atmosphere packaging on radiation tolerance in the phytosanitary pest melon fly (Diptera: Tephritidae).

Modified atmosphere packaging (MAP) produces a low-oxygen (O2) environment that can increase produce shelf life by decreasing product respiration and growth of pathogens. However, low O2 is known to increase insect tolerance to irradiation, and the use of MAP with products treated by irradiation before export to control quarantine pests may inadvertently compromise treatment efficacy. Melon fly, Bactrocera cucurbitae Coquillet (Diptera: Tephritidae), is an important economic and quarantine pest of tropical fruits and vegetables, and one of the most radiation-tolerant tephritid fruit flies known. The effect of low O2 generated by MAP on the radiation tolerance of B. cucurbitae was examined. Third-instar larval B. cucurbitae were inoculated into ripe papayas and treated by 1) MAP + irradiation, 2) irradiation alone, 3) MAP alone, or (4) no MAP and no irradiation, and held for adult emergence. Three types of commercially available MAP products were tested that produced O2 concentrations between 1 and 15%, and a sublethal radiation dose (50 Gy) was used to allow comparisons between treatments. Ziploc storage bags (1-4% O2) increased survivorship to adult from 14 to 25%, whereas Xtend PP61 bags (3-8% O2) and Xtend PP53 bags (11-15% O2) did not enhance survivorship to the adult stage in B. cucurbitae irradiated at 50 Gy. Radiation doses approved by the United States Department of Agriculture and the International Plant Protection Commission for B. cucurbitae and Ceratitis capitata (Wiedemann) (Mediterranean fruit fly) are 150 and 100 Gy, respectively. In large-scale tests, 9,000 B. cucurbitae and 3,800 C. capitata larvae infesting papayas in Ziploc bags were irradiated at 150 and 100 Gy, respectively, with no survivors to the adult stage. MAP can increase insect survivorship during irradiation treatment at certain doses and O2 concentrations, but should not compromise the efficacy of the 150-Gy generic radiation treatment for tephritid fruit flies or the 100-Gy radiation treatment for C. capitata.

Cold storage enhances the efficacy and margin of security in postharvest irradiation treatments against fruit flies (Diptera: Tephritidae).

Cold storage is used to preserve fruit quality after harvest during transportation in marketing channels. Low temperature can be a stressor for insects that reduces survivorship, and cold storage may contribute to the efficacy of postharvest quarantine treatments such as irradiation against quarantine insect pests. The combined effect of irradiation and cold storage was examined in a radiation-tolerant fruit fly, Bactrocera cucurbitae Coquillet (melon fly), and a radiation-intolerant fruit fly, Ceratitis capitata (Wiedemann) (Mediterranean fruit fly) (Diptera: Tephritidae). Third instars on diet or in papaya were treated with a sublethal radiation dose of 30 Gy and stored at 4 or 11 degrees C for 3-13 d and held for adult emergence. For both fruit fly species, survival of third instars to the adult stage generally decreased with increasing cold storage duration at 4 or 11 degrees C in diet or papaya. Survivorship differences were highly significant for the effects of substrate (diet > papaya), temperature (11 > 4 degrees C),and irradiation (0 > 30 Gy). Few Mediterranean fruit flies survived in any cold storage treatment after receiving a radiation dose of 30 Gy. No melon fly larvae survived to the adult stage after irradiation and 11 d cold storage at 4 or 11 degrees C in papayas. Cold storage enhances the efficacy and widens the margin of security in postharvest irradiation treatments. Potentially irradiation and cold storage can be used in combination to reduce the irradiation exposure requirements of quarantine treatments.

Photosensitizing effect of hematoporphyrin IX on immature stages of Ceratitis capitata (Diptera: Tephritidae).

Immature stages of Ceratitis capitata were tested as a model for hematoporphyrin IX (HP IX) phototoxicity. The lethal concentration 50 (LC(50)) of HP IX in the food was determined during postembryonic development until adult emergence as 0.173 mm (95% CI: 0.138-0.209). The corresponding HP IX LC(50) during the dispersal period alone was 0.536 mm (95% CI: 0.450-0.633). HP IX toxicity was compared against Phloxine B (PhB) (0.5 mm). HP IX elicited a mortality of 90.87%, which was mainly concentrated during prepupal and early pupal stages. PhB mortality was much lower (56.88%) and occurred mainly during the adult pharate stage. A direct correlation between light-dependent HP IX mortality, evidence of reactive oxygen species (ROS) and lipid peroxidation (conjugated dienes and thiobarbituric acid reactive substances) was established in C. capitata larvae. ROS were found to be very significant in both the brain and in the gut.

A new generation of X ray irradiators for insect sterilization.

Recent fears of terrorism have provoked an increase in delays and denials of transboundary shipments of radioisotopes. This represents a serious constraint to sterile insect technique (SIT) programs around the world as they rely on the use of ionizing radiation from radioisotopes for insect sterilization. To validate a novel X ray irradiator, a series of studies on Ceratitis capitata (Wiedemann) and Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) were carried out, comparing the relative biological effectiveness (RBE) between X rays and traditional gamma radiation from 60Co. Male C. capitata pupae and pupae of both sexes of A. fraterculus, both 24-48 h before adult emergence, were irradiated with doses ranging from 15 to 120 Gy and 10-70 Gy, respectively. Estimated mean doses of 91.2 Gy of X and 124.9 Gy of gamma radiation induced 99% sterility in C. capitata males. Irradiated A. fraterculus were 99% sterile at approximately 40-60 Gy for both radiation treatments. Standard quality control parameters and mating indices were not significantly affected by the two types of radiation. The RBE did not differ significantly between the tested X and gamma radiation, and X rays are as biologically effective for SIT purposes as gamma rays are. This work confirms the suitability of this new generation of X ray irradiators for pest control programs that integrate the SIT.

Manipulation of the microbiota of mass-reared Mediterranean fruit flies Ceratitis capitata (Diptera: Tephritidae) improves sterile male sexual performance.

The sterile insect technique (SIT) is a method of biological control whereby millions of factory reared sterile male insects are released into the field. This technique is commonly used to combat the Mediterranean fruit fly (Ceratitis capitata, Diptera: Tephritidae). Sterile medfly males are less competent in attracting and mating with wild females, a property commonly linked to the irradiation process responsible for the sterilization. As bacteria are important partners in the fly's life cycle, we used molecular analytical methods to study the community structure of the gut microbiota in irradiated male medflies. We find that the sterilizing irradiation procedure affects the gut bacterial community structure of the Mediterranean fruit fly. Although the Enterobacteriaceae family remains the dominant bacterial group present in the gut, the levels of Klebsiella species decreases significantly in the days after sterilization. In addition, we detected substantial differences in some bacterial species between the mass rearing strain Vienna 8 and the wild strain. Most notable among these are the increased levels of the potentially pathogenic species Pseudomonas in the industrial strain. Testing the hypothesis that regenerating the original microbiota community could result in enhanced competitiveness of the sterile flies, we found that the addition of the bacterial species Klebsiella oxytoca to the postirradiation diet enables colonization of these bacteria in the gut while resulting in decreased levels of the Pseudomonas sp. Feeding on diets containing bacteria significantly improved sterile male performance in copulatory tests. Further studies will determine the feasibility of bacterial amelioration in SIT operations.

Conditional embryonic lethality to improve the sterile insect technique in Ceratitis capitata (Diptera: Tephritidae).

BACKGROUND: The sterile insect technique (SIT) is an environment-friendly method used in area-wide pest management of the Mediterranean fruit fly Ceratitis capitata (Wiedemann; Diptera: Tephritidae). Ionizing radiation used to generate reproductive sterility in the mass-reared populations before release leads to reduction of competitiveness. RESULTS: Here, we present a first alternative reproductive sterility system for medfly based on transgenic embryonic lethality. This system is dependent on newly isolated medfly promoter/enhancer elements of cellularization-specifically-expressed genes. These elements act differently in expression strength and their ability to drive lethal effector gene activation. Moreover, position effects strongly influence the efficiency of the system. Out of 60 combinations of driver and effector construct integrations, several lines resulted in larval and pupal lethality with one line showing complete embryonic lethality. This line was highly competitive to wildtype medfly in laboratory and field cage tests. CONCLUSION: The high competitiveness of the transgenic lines and the achieved 100% embryonic lethality causing reproductive sterility without the need of irradiation can improve the efficacy of operational medfly SIT programs.

Effects of gamma-irradiation on midgut proteolytic activity of the mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae).

The Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann), is a key pest of citrus in Spain because of significant yield losses and to quarantine restrictions. Biologically based control methods, such as the Sterile Insect Technique (SIT), which relies on the sterilization by irradiation of large numbers of insects, is gaining an increasing role in the control of medfly in Mediterranean areas. However, gamma-irradiation might damage the midgut epithelium cells, causing a lowering of nutritive assimilation that can negatively affect adult performance. Irradiation effects on digestive physiology are well established for a number of insect pests, but there is no information on medfly. Our aim was to determine the effects of gamma-irradiation on C. capitata digestive protease activity. Both larvae and adults were found to use a similar proteolytic system based on aspartyl-, trypsin-, chymotrypsin-, amino peptidase-, and carboxypeptidase A- and B-like activities. Pupae of the Vienna-7 (tsl) strain were irradiated at 70 or 140 Gy, two days before emergence, and the adults fed during 5 days on sugar-protein (4:1) diets. Protease activity was measured in midgut extracts and compared with males non-irradiated reared in the same conditions. The results showed that the irradiation doses tested had no effect on the digestive proteolytic activities of medfly adults. Moreover, the longevity of irradiated medflies at the highest dose (140 Gy) was similar to that of controls.

Effects of irradiation on Mediterranean fruit flies (Diptera: Tephritidae): emergence, survivorship, lure attraction, and mating competition.

Irradiation of puparia in Mediterranean fruit fly, Ceratitis capitata (Wiedemann), sterile insect release programs can negatively affect adult fly performance. Emergence, survivorship, lure attraction, and mating competition tests were performed on irradiated and unirradiated Mediterranean fruit flies in Hawaii. Unirradiated flies of the Vienna-7 (tsl) strain had higher emergence, flight ability, and survivorship compared with irradiated flies. In general, unirradiated flies were more responsive to trimedlure, but this effect was not consistent for all strains at every age. Laboratory strains, of both unirradiated and irradiated flies, responded to trimedlure at a younger age than wild flies, which may be a result of inadvertent selection for decreased development time in laboratory-reared flies. Mating competition tests with irradiated and unirradiated flies showed no significant differences. Costs associated with the irradiation process and the development of alternative control techniques are discussed.

Genetic stability of sexing strains based on the locus sw of Ceratitis capitata.

This report deals with the process of improving the stability of medfly, Ceratitis capitata, genetic sexing strains (GSS) based on the sw mutation on chromosome 2. This gene affects the rate of development as well as the eye colour and iridescence. The improved sexing strains were produced by mapping sw with deletions and then inducing and screening for new translocations with breakpoints close to the marker. The stability was assessed in large populations over many generations. Twenty-two new Y-2 translocations were identified and polytene chromosome analysis was performed to locate breakpoints. The translocation strains were ranked according to the distance of their breakpoints from sw. The map position of sw is region 20D on 2R. As data on the stability of the 22 strains accumulated, Cast 191 was shown to be the most promising as no recombination between sw and the male sex was found. After rearing the strain for 22 generations under semi-mass rearing conditions, with a population size of 15,000 adults and scoring 1000 flies per generation, only one such event was detected (estimated frequency = 3.1 x 10(-6)). Further tests are being carried out with this strain to assess its suitability as a genetic sexing strain for medfly Sterile insect technique (SIT).

Mass rearing of temperature sensitive genetic sexing strains in the Mediterranean fruit fly (Ceratitis capitata).

Genetic sexing strains (GSS) based on the temperature sensitive lethal (tsl) mutation are being used to produce sterile male medflies for large scale sterile insect technique (SIT) programmes for this pest. The use of male-only strains increases the overall efficiency of the technique. Currently more than 1.4 billion sterile male-only pupae are produced per week in different facilities around the world. Due to the mutations used to construct these strains, that is, translocations and selectable markers, they require different and more careful mass rearing procedures than do bisexual strains (BSS). The basic rearing technology has been developed and can be used to produce only males on a predictable basis to a level of 99.9% accuracy. If specific rearing procedures are followed, then tsl-based GSS has a rearing efficiency that is equal to that of a BSS and it is already know that males produced by the tsl-based GSS are of equal quality to males produced by BSS. Based on current rearing technology the cost of production of male pupae is about the same for both types of strain. This is due to the large colony that is required for the tsl-based GSS. This paper discusses the considerations that need to be taken into account during mass rearing of GSS and identifies the most efficient production processes that are currently available.

Generic ionizing radiation quarantine treatments against fruit flies (Diptera: Tephritidae) proposed.

Tephritid fruit flies comprise the most important group of quarantined pests of fresh produce. Most quarantine treatments of fresh agricultural commodities are directed against these pests, and considerable effort in detection, trapping, and population control is expended worldwide to prevent these pests from invading new territories. Ionizing radiation has been studied for 70 yr for its possible use as a quarantine treatment against fruit flies, but has only been applied commercially on a limited basis since 1995. The treatment has great potential and will probably be used extensively in the future as it is tolerated by more species of fruits than any other major treatment. The U.S. Department Agriculture, Animal and Plant Health Inspection Service only recently proposed allowing irradiation for fresh agricultural imports from other countries, and other countries are studying proposals to do likewise. In 1991, the International Consultative Group on Food Irradiation recommended a generic dose against all tephritid fruit flies of 150 Gy. This article examines the literature dealing with irradiation quarantine treatments against fruit flies and recommends minimum absorbed doses of 70 Gy for Anastrepha spp., 101 Gy for Bactrocera jarvisi and B. tryoni, and 150 Gy for all Tephritidae except when fruits have been stored in hypoxic atmospheres.