Irradiated Larvae Research Articles

Impact of Pulsed Laser Irradiations from Nd:YAG Laser on the Larvae of the Fouling Barnacle Balanus amphitrite

The environmental hazards originating from the persistent use of antifouling agents containing toxic chemicals are alarming. Therefore, methods that do not rely on toxic elements are wanted. The effectiveness of pulsed laser irradiations from an Nd:YAG laser to induce damage to the larvae of a fouling barnacle, Balanus amphitrite, was evaluated in the laboratory. The average fluence tested was 0.1 J cmm 2. Three larval stages, viz. nauplii stage II and stage IV and cyprid larvae were exposed to laser irradiations for 2 s, 10 s, 30 s, 1 min, 5 min and 15 min in the case of nauplii stage II, and 2, 10 and 30 s in case of nauplii stage IV and cyprid larvae. Mortality occurred immediately after, as well as 1 and 3 d after the irradiation, and the settlement rate of cyprid larvae was investigated in the laboratory. Mortality in stage II, stage IV and cyprid larvae immediately after 2 s irradiation was 30.1 ± 0.5%, 9.3 ± 0.2 and 16.7 ± 4.8, respectively. Mortality after 3 d however showed higher values, viz. 84.4 ± 6.7%, 50 ± 4%, and 75 ± 9.4 in stage II, stage IV and cyprid larvae, respectively. Mortality increased with the period of laser irradiation. The stage II nauplii showed 100% mortality after 10 s irradiation. The mortality observed in stage IV and cyprid larvae was less than that found for the stage II nauplii. The IV stage larvae which survived could successfully moult to the next stage, whilst the irradiated cyprid larvae could not settle. The results thus showed that pulsed low power laser irradiations could cause significant damage to the larvae of this common fouling barnacle.

B-cell deficiency suppresses vaccine-induced protection against murine filariasis but does not increase the recovery rate for primary infection.

To establish the role of B cells and antibodies in destroying filariae, mice lacking mature B cells and therefore unable to produce antibodies were used. Litomosoides sigmodontis offers a good opportunity for this study because it is the only filarial species that completes its life cycle in mice. Its development was compared in B-cell-deficient mice (BALB/c muMT mice) and wild-type BALB/c mice in two different in vivo situations, vaccination with irradiated larvae and primary infection. In all cases, mice were challenged with subcutaneous inoculation of 40 infective larvae. Vaccine-induced protection was suppressed in B-cell-deficient mice. In these mice, eosinophils infiltrated the subcutaneous tissue normally during immunization; however, their morphological state did not change following challenge inoculation, whereas in wild-type mice the percentage of degranulated eosinophils was markedly increased. From this, it may be deduced that the eosinophil-antibody-B-cell complex is the effector mechanism of protection in vaccinated mice and that its action is fast and takes place in the subcutaneous tissue. In primary infection, the filarial survival and growth was not modified by the absence of B cells. However, no female worm had uterine microfilariae, nor did any mice develop a patent infection. In these mice, concentrations of type 1 (gamma interferon) and type 2 (interleukin-4 [IL-4], IL-5 and IL-10) cytokines in serum were lower and pleural neutrophils were more numerous. The effects of the muMT mutation therefore differ from those in B1-cell-deficient mice described on the same BALB/c background, which reveal a higher filarial recovery rate and microfilaremia. This outlines B2-cell-dependent mechanisms as favorable to the late maturation of L. sigmodontis.

Expression of cell surface adhesion molecules by peripheral blood eosinophils during Trichostrongylus colubriformis infection in sheep.

The effect of infection of sheep with the gastrointestinal nematode parasite Trichostrongylus colubriformis on expression of adhesion molecules CD11a, CD11b, CD11c, CD18, CD44, CD49d and CD62L by peripheral blood eosinophils was examined by flow cytometry. Initially, to establish the sensitivity of adhesion molecules to inflammatory signals, eosinophil-rich exudates were elicited in non-lactating mammary glands of immune sheep by infusion of 50 microg of soluble antigen extract from T. colubriformis third stage larvae. Eosinophils comprised 40.8% of mammary leucocytes and 4.5% of peripheral blood leucocytes. In comparison with blood, the percentage of eosinophils expressing CD18 increased and the percentage expressing CD62L decreased in exudates and the mean fluorescent intensity, an indicator of receptor number per cell, for CD11a and CD49d also decreased on exudate eosinophils. Peripheral blood eosinophils were examined over 8 weeks during trickle infection of immune sheep with infective or irradiated third stage larvae of T. colubriformis. During the last 3 weeks of infection, CD11a staining decreased in infected sheep and CD44 staining decreased in sheep receiving either infective or irradiated larvae. Other surface markers did not change. The results indicate that systemic changes in expression of adhesion molecules by eosinophils occur during T. colubriformis infection in sheep.

Selection of sheep for response to Trichostrongylus colubriformis larvae: genetic parameters

AbstractData were analysed from a long-term selection experiment with Merino sheep, based on immunological responsiveness to the intestinal nematode Trichostrongylus colubriformis. For the first 14 years of selection, the criterion was the mean of five fortnightly faecal worm-egg counts (FECs) of pen-housed lambs that were vaccinated with irradiated larvae then challenged with normal T. colubriformis larvae. For most of the lambs born in the subsequent 6 years, the selection criterion was the mean of three weekly FECs of grazing lambs following a secondary challenge with T. colubriformis larvae. Data from 2233 lambs were included in the analyses.At the end of the experiment, the lines selected for high and low response to challenge differed by 2·3 to 2·9 phenotypic standard deviations. The heritability of average pen-tested FEC was 0·38 (s.e. 0·04), similar to that for average FEC after secondary field challenge (0·37, s.e. 007). Average FECs from the primary field challenge were less heritable (0·21, s.e. 006). Counts recorded on pen-tested animals at 3, 5, 7, 9 and 11 weeks after infection were all highly correlated genetically (estimates 0·93 to 0·99) and of similar heritability (0·33 to 0·39) with a non-significant tendency for later FECs to be more heritable. Phenotypic correlations were lower, ranging from 0·60 for counts 8 weeks apart, to 0·78 to 0·81 for adjacent samples 2 weeks apart. Single-record heritability estimates at 3, 4 and 5 weeks after secondary infection in grazing lambs did not differ statistically from each other but were highest at the 5th week after infection at 0·33 (s.e. 007).FECs determined in pens were imperfectly correlated with FECs determined at pasture (genetic correlation 0·72, s.e. 013). Phenotypic correlations of single-record FECs across primary and secondary field challenges were low (around 0·2), although the genetic correlation between mean primary FEC and mean secondary FEC was not significantly different from unity. Testing under standardized conditions in pens did not result in consistently higher heritabilities than testing immunologically primed lambs at pasture. Repeated measurement in a breeding programme offers little additional benefit, except when lambs have had ample prior experience of parasite infection, with the two measures separated by an anthelmintic treatment. If an objective of a Merino breeding programme is to reduce FEC under field conditions, then using one measure in the field, approximately 3 to 5 weeks after a secondary artificial infection, is likely to lead to long-term progress.

IL-5 is essential for vaccine-induced protection and for resolution of primary infection in murine filariasis.

The pathways conferring immunity to human filariases are not well known, in part because human-pathogenic filariae do not complete a full life cycle in laboratory mice. We have used the only fully permissive infection of mice with filariae, i.e., infection of BALB/c mice with the rodent filarial nematode Litomosoides sigmodontis. Our previous results showed that worm development is inversely correlated with Th2 cytokine production and eosinophilia. The scope of the present study was to directly elucidate the role of interleukin-5 (IL-5) and eosinophils in controlling the development of L. sigmodonitis after vaccination and in primary infection. BALB/c mice immunized with irradiated third-stage larvae (L3) were confirmed to have elevated IL-5 levels as well as high subcutaneous eosinophilia and to attack and reduce incoming larvae within the first 2 days, resulting in 70% reduction of worm load. Treatment of vaccinated mice with anti-IL-5 antibody (TRFK-5) suppressed both blood and tissue eosinophilia and completely abolished protection. This demonstrates, for the first time in a fully permissive filarial infection, that IL-5 is essential for protection induced by irradiated L3 larvae. In contrast, in primary-infected mice, anti-IL-5 treatment did not modify filarial infection within the 1st month, most likely because during primary infection IL-5-dependent mechanisms such as subcutaneous eosinophilia are induced too late to disturb worm establishment. However, there is a role for IL-5 late in primary infection where neutrophil-dependent worm encapsulation is also under the control of IL-5.

Vaccination of young lambs against infection with Nematodirus battus using gamma irradiated larvae

Helminthologically naÏve 6-week-old Suffolk lambs were given 1–3 doses of 20 000 γ-irradiated infective larvae (L3) of the nematode Nematodirus battus at weekly intervals. Following an anthelmintic drench they were challenged with 50 000 viable L3 at 10 weeks of age. Nematode worm burdens 14 days post-challenge showed a significant (P<0.01) 66% reduction in the single vaccine dose group. The two and three dose groups had mean worm burdens which were 30 and 42% lower than controls, respectively, although these were not statistically significant. There was little measurable stimulation of the immune system in the vaccinated lambs, suggesting that the repeatedly dosed animals may have developed immunological unresponsiveness to the parasite.

Host protective immunity and vaccine development studies in lymphatic filariasis

Lymphatic filariasis caused mainly by infection fromWuchereria bancrofti andBrugia malayi remains as the major cause of clinical morbidity in tropical and subtropical countries. Development of vaccine against filarial infection can act as additional measure to the existing therapeutic and vector control methods in the control of this disease. The main hurdles in the development of anti-filarial vaccine are the strict primate specificity ofWuchereria bancrofti, the paucity of parasite material, the diversity of clinical manifestations and their associated complex immune responses, lack of clear understanding on host-parasite interactions and the mechanisms involved in protective immunity. However in the past few years, the information generated in immuno-epidemiological studies, correlated with observations in experimental animals suggests that a filarial vaccine is feasible. Initially live irradiated infective larvae have been successfully used to induce high level of protective immunity in several animal models. Applying diverse strategies, variety of purified or recombinant filarial antigens have been explored for their ability to induce protection in different host-parasite systems. Some of these targeted filarial antigens induced high level of resistance in experimental animals against challenge infections. More focussed studies on thorough characterization of parasitological and immunological changes associated with resistance induced by such candidate protective antigens and on delivery mechanisms and safety aspects will be crucial in their selection for possible use in humans.

Drastic reduction of a filarial infection in eosinophilic interleukin-5 transgenic mice.

In order to establish the role of eosinophils in destroying parasites, transgenic mice have been used in experimental helminthiases but not in filariasis. Litomosoides sigmodontis offers a good opportunity for this study because it is the only filarial species that completes its life cycle in mice. Its development was compared in transgenic CBA/Ca mice overexpressing interleukin-5 (IL-5) and in wild-type mice following subcutaneous inoculation of 40 infective larvae. An acceleration of larval growth was observed in the IL-5 transgenic mice. However, the recovery rate of adult worms was considerably reduced in these mice, as evidenced 2 months postinoculation (p.i.). The reduction occurs between days 10 and 30 p.i. in the coelomic cavities. As early as day 10, spherical aggregates of eosinophils and macrophages are seen attached on live developing larvae (always similarly localized on the worm) in both wild-type and transgenic mice. However, on day 60 p.i., granulomas were found in the transgenic mice only, probably because of the higher density of eosinophils. Furthermore, on day 30 p.i., young filariae are seen trapped in granulomas, some of them surrounded by Splendore-Hoeppli deposits, which illustrates the release of the major basic protein by eosinophils. The high protection rate obtained (65%) is similar to that observed previously in BALB/c mice following vaccination with irradiated larvae. Both protocols have a common factor, the high production of IL-5 and eosinophilia. However, protection occurs later in primary infected transgenic mice because specific antibodies are not yet present at the time of challenge.

Interleukin-5 is essential for vaccine-mediated immunity but not innate resistance to a filarial parasite.

The study of protective immune mechanisms effective against filarial nematodes has been hampered by the inability of these important human pathogens to infect laboratory mice. Recently, Litomosoides sigmodontis, a natural parasite of rats, has been developed as a valuable model for the study of filarial infection. BALB/c mice are fully susceptible to infection with L. sigmodontis third-stage larvae and develop patent infection. In contrast, mice on the C57BL background are resistant, and parasites undergo only a single molt and do not mature to adulthood. We used interleukin-5 (IL-5)-deficient mice on the C57BL/6 background to address the role of IL-5 and eosinophils in the innate resistance of C57BL/6 mice. We found no differences in parasite survival between IL-5-deficient and C57BL/6 mice. However, when these mice were used for the analysis of vaccine-mediated immunity, a critical role for IL-5 was elucidated. Mice genetically deficient in IL-5 were unable to generate a protective immune response when vaccinated with irradiated larvae, whereas C57BL/6 mice were fully protected from challenge infection. These studies help to clarify the highly controversial role of eosinophils in filarial infection.

Onchocerca ochengi infections in cattle as a model for human onchocerciasis: recent developments

The bovine parasite Onchocerca ochengi is a nodule-dwelling filarial nematode, closely related to O. volvulus, the causal agent of human River Blindness, and, sharing with it, the same vector. This brief review, based on a presentation at the BSP Autumn Symposium 1999, describes recent work supported by the WHO Drug Development Research Macrofil programme and the Edna McConnell Clark Foundation vaccine development programme, to research the chemotherapy and immunology of onchocerciasis utilising this model system, with experimental infections in Liverpool and field infections in northern Cameroon. In a series of chemotherapeutic trials involving 10 compounds in 20 treatment regimes, the comparability of drug efficacy against O. ochengi with that described against O. volvulus has been demonstrated. Repeated, long-term treatment with oxytetracycline has been shown to be macrofilaricidal and the effect is hypothesized to be related to action on Wolbachia endobacteria, abundant in O. ochengi. Avermectins/milbemycins are not macrofilaricidal (even in high and repeated long-term treatments) but induce sustained abrogation of embryogenesis. In prospective, field exposure experiments with naive calves, prophylactic treatments with ivermectin and moxidectin prevented the development of adult worm infection, raising the possibility that drug-attenuated larval challenge infections may induce immunity. Putatively immune adult cattle exist in endemically exposed populations, and these have been shown to be significantly less susceptible to challenge than age-matched naive controls, whereas radically drug-cured, previously patently-infected cattle were not. Experimental infections with O. ochengi have revealed the kinetics of the immune response in relation to parasite development and demonstrate analogous responses to those reported in O. volvulus infection in humans and chimpanzees. In an immunization experiment with irradiated L3 larvae, cattle were significantly protected against experimental challenge--the first such demonstration of the experimental induction of immunity in a natural Onchocerca host-parasite system. Taken collectively, these studies not only demonstrate the similarity between the host-parasite relationships of O. ochengi in cattle and O. volvulus in humans, but promise to advance options for the control of human onchocerciasis.

Cellular responses to Loa loa experimental infection in mandrills (Mandrillus sphinx) vaccinated with irradiated infective larvae.

In order to shed light on the mechanisms of antifilarial protective immunity, we investigated the course of experimental loaiosis after vaccination in a nonhuman primate host, Mandrillus sphinx. Six vaccinated (V) mandrills received 50 irradiated L3 while six nonvaccinated (NV) received saline solution on days -60, -30 and -15. All animals were challenged with 100 intact L3 (day 0). Parasitological and immunological status were followed for 9 months. Vaccination delayed the appearance and mean peak of microfilaraemia. Five mandrills (Mf-) were never microfilaraemic (one V mandrill) or microfilaraemic on only one occasion (2 V and 2 NV), the other seven having stable microfilaraemia (Mf+). The cytokine response of peripheral blood mononuclear cells to L3 (L3 Ag) was Th2 dominated, while microfilariae (Mf Ag) elicited a Th0-like response. During vaccination, Th2 cytokine production significantly increased in V mandrills against L3 Ag, as well as Mf Ag, whereas Th1 cytokines decreased. On day 60 postinoculation, cellular proliferation was higher in V mandrills in response to L3 and Mf Ags and PHA-L mitogen. At the end of prepatency (on day 130), mandrills with delayed appearance of microfilaraemia exhibited a high, transient IL-2 and IL-4 secretion in response to L3 Ag. Finally, high anti-Mf Th2 cytokine levels characterized Mf-mandrills not only during prepatency, but also (for IL-5) before immunization. However, the presence of a balanced Th1 anti-L3 response during prepatency in the amicrofilaraemic mandrill suggests its importance in protective immunity. Taken together, our data suggest that Th2 cells and also Th1 components of the antifilarial response, especially to larval antigen, may contribute to parasite elimination.

Somatic cell mutation and photoproduct formation inDrosophila induced by monochromatic UV light in sunlight

We recently reported that natural sunlight can induce somatic mutation and chromosomal recombination in Drosophilia, as detected by the wing spot test. Simultaneously, cyclobutane thymine dimers (CTDs) and (6-4)photoproducts [(6-4)PPs] are formed in the DNA. In the present work, we have performed monochromatic UV irradiation for evaluating the action spectrum of sunlight in genotoxicity. Third instar larvae in petri dishes were irradiated by monochromatic UV light at a wavelength between 310 and 360 nm. The mutant spots induced by the irradiation of 310, 320, 330, and 340 nm light were (4.4±1.0)×10−1, (1.1±0.2)×10−2, (2.5±0.7)×10−3, and (2.0±0.7)×10−3 spots/wing/kJ/m2, respectively. The mutagenicity at 360 nm irradiation was not significant. The amounts of photoproducts in irradiated larval DNA were measured by an enzyme-linked immunosorbent assay (ELISA) using the monoclonal antibodies against CTDs and (6-4)PPs. CTDs were found in the DNA from all of these irradiated larvae, and their amounts increased with the UV dose: the longer the wavelength, the lower the CTD formation and the mutagenesis. These data suggest that, in the sunlight, the component around 310–340 nm causes the somatic cell mutation by forming thymine dimers as main lesions. © 2000 John Wiley & Sons, Inc. Environ Toxicol 15: 496–499, 2000

Efficacy of gamma irradiation against Sitophilus granarius (L.) (Coleoptera: Curculionidae)

The lethal and sterilizing responses to gamma irradiation of eggs, larvae, pupae, and three-day- and four-week-old adults of Sitophilus granarius were investigated. Doses were 0, 10, 30, 50, 70, 100, 300 and 500 Gy. Eggs and larvae were unable to develop to adults following doses of 30–500 Gy. Emergence of adults from irradiated eggs and larvae occurred at a dose of 10 Gy. Pupae developed to the adult stage following doses of 10–70 Gy. A dose of 70 Gy at the pupal and four-week-old adult stages caused sterility. Three-day-old adults were most tolerant of irradiation and required 100 Gy for sterility. The percentage of damage to wheat grains caused by pre-adult stages following various irradiation doses was determined.

In the absence of IL-12, the induction of Th1-mediated protective immunity by the attenuated schistosome vaccine is impaired, revealing an alternative pathway with Th2-type characteristics.

Vaccination of mice with irradiated Schistosoma mansoni larvae confers high levels of immunity which is mediated by Th1-type lymphocytes. To investigate a possible role for IL-12 in the induction of protection, we have compared the immune response of IL-12 p40-deficient (KO) mice and their C57BL/6 (WT) counterparts following vaccination. Cultured lymph node cells from KO mice had markedly altered cytokine profiles with significantly decreased production of IFN-gamma increased IL-4. Correspondingly, KO mice had enhanced levels of IgE. After challenge, cells recovered from the lungs of KO mice secreted abundant IL-4 and IL-5 but little IFN-gamma, while flow cytometric and histological analysis of lung cell populations recorded a very high proportion of eosinophils. The levels of protection in KO mice were substantially lower than in their WT counterparts, demonstrating the importance of IL-12 and Th1-mediated immune responses. This conclusion is reinforced by the administration of rIL-12 to KO mice immediately after vaccination which led to increased IFN-gamma and the restoration of protective immunity. Nevertheless, the data also indicated that the limited levels of protection induced in KO mice occur via an IL-12-independent pathway, possibly mediated by Th2 cells.

Cellular immune response to Loa Loa experimental infection in mandrills vaccinated with irradiated infective larvae

Cellular immune response to Loa Loa experimental infection in mandrills vaccinated with irradiated infective larvae

Immune responses to third stage larvae of Onchocerca volvulus in interferon-gamma and interleukin-4 knockout mice.

To shed clarity on the dichotomy of reported results relative to the significance of T helper-1 vs T helper-2 immune responses in onchocerciasis, we compared the survivability of Onchocerca volvulus third-stage larvae (L3) in immunized mice that had either a targeted disruption of the Interleukin-4 or Interferon-gamma gene. Treatment groups consisted of control mice and mice immunized with irradiated O. volvulus L3. All mice were challenged with diffusion chambers containing viable L3. Vaccinated IL-4-/- were unable to kill this larval target. In contrast, vaccinated INF-gamma-/- and C57BL/6 mice, exhibited high levels of killing, had elevated levels of IL-4 and significantly greater numbers of eosinophils in their diffusion chambers than the IL-4-/-. Whereas, levels of IFN-gamma in all three groups of immunized mice were equivalent to those of control mice, levels of IL-5 were elevated, even in the IL-4-/-, indicating that cytokines other than IL-4 were involved in its production. The protective immune response to third-stage larvae of O. volvulus in mice vaccinated with irradiated larvae has an absolute IL-4 requirement.

Reduction of Challenge Parasite Population in Gerbils, Meriones unguiculatus, Chronically Infected with Schistosoma mansoni

Homologous challenge parasite attrition in gerbils, Meriones unguiculatus, chronically infected with Schistosoma mansoni, was investigated by the recovery technique of retrograde portal perfusion. Whereas the animals vaccinated with gamma- or UV-attenuated cercariae of S. mansoni showed a marginal level of resistance, the chronically infected gerbils exhibited significant parasite attrition against a homologous challenge infection. Our data do not corroborate a previous report suggesting a lack of resistance in gerbils with chronic S. mansoni infection. The gerbil represents an additional experimental host for evaluating acquired resistance to S. mansoni, including that induced by previous exposure to irradiated larvae on chronic infection.

Acanthocheilonema viteae: vaccination with irradiated L3 induces resistance in three species of rodents (Meriones unguiculatus, Mastomys coucha, Mesocricetus auratus).

Three species of rodents were immunized with 50 irradiated (35 krad) stage-3 larvae (L3) of the filaria Acanthocheilonema viteae and challenged with an infection of normal L3. The immunization induced a significant reduction of the worm burden developing from the challenge infection in all host species, the jird (Meriones unguiculatus), the multimammate rat (Mastomys coucha) and the golden hamster (Mesocricetus auratus). The induced resistance was highest in jirds (92.5 +/- 9.7) followed by golden hamsters (59.4 +/- 26.6) and multimammate rats (55.1 +/- 40.4). The time course of antibody response against antigens of L3, adult worms and microfilariae, as studied by ELISA, showed quantitative and qualitative differences between the species. The antibody response against L3 antigens in immunoblots was similar in all species. Only one of the golden hamsters developed an antibody response against the surface of vector derived L3, while sera of jirds and multimammate rats did not react with L3 surface.

Identification of larval-stage-specific antigens of Onchocerca volvulus uniquely recognized by putative immune sera from humans and vaccination sera from animal models.

Immunity to Onchocerca volvulus is indicated by the existence of putative immune individuals (PI), who do not develop patent infections or clinical symptoms after living in endemic areas for extended periods of time. To gain insight into the nature of the humoral response of PI from Liberia and Ecuador, their sera and those of infected individuals were investigated and compared using western blots. The antigen preparations used were extracts of third- and fourth-stage larvae (L3 and L4, respectively), excretory-secretory proteins (ES-L3) and extracts of third-stage larvae (L3-day2) that had been cultured for 2 days, and extracts of nodular and skin microfilariae. Whereas some antigens were commonly recognized by all individuals, many stage-specific antigens (of 68, 60, 55, 46, 43, 42, 20 and 18 kDa in L3; 100, 88, 80, 50, 48, 45, 40 and 20 kDa in L3-day2; 110, 80 and 72 kDa in ES-L3; 48, 18, 16 and 14 kDa in L4; and 115, 60, 47, 43 and 17 kDa in skin and/or nodular microfilariae) were uniquely recognized by sera from PI. Some of the unique antigens were also recognized by sera from mice and a chimpanzee that were resistant to challenge after immunization with irradiated third-stage larvae. The results support the validity of the assumption that a unique status of immunity develops in certain individuals living in areas where onchocerciasis is endemic.

Gamma radiation as a quarantine treatment for the Mediterranean fruit fly (Diptera:Tephritidae).

Effects of gamma radiation on the egg and larval stages of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), were examined. Eggs and larvae were exposed at different ages to a series of gamma radiation doses ranging from 5 to 1,280 Gy. Eggs in the 1st half of their development (1 and 24 h) were very sensitive to irradiation treatment (20 Gy prevented egg hatch). However, mature eggs (48 h old) were much more tolerant. When mature eggs were irradiated, a dose of 640 Gy prevented egg hatch. Pupariation of eggs treated at 1 and 24 h was significantly affected at a dose of 20 Gy; adult emergence of eggs at 1, 14, and 48 h was prevented at this dose. The larval stage was significantly more resistant to radiation than the egg stage. Survival to the pupal stage increased with increasing age of larvae, and decreased with increasing dose. The minimum dose required to prevent pupariation ranged from over 160 Gy for the 1st instar to > 640 Gy for mature 3rd instars. In contrast, doses required to prevent adult emergence from irradiated larvae were relatively low and ranged from 10-20 to 20-40 Gy depending on the age of the insects when irradiated. Tests in which > 100,000 mature larvae were treated in air with a dose of 40 Gy resulted in no adult emergence. Similar results were obtained when 3rd instars were irradiated inside natural host fruits in a small scale laboratory experiment.