Irradiation of Anastrepha obliqua (Diptera: Tephritidae) revisited: optimizing sterility induction

The effects of irradiation doses increasing from 0 to 100 Gy (1 Gy is energy absorbed in J kg(-1) of irradiated material) on fertility, flight ability, survival, and sterile male mating performance were evaluated for mass-reared Anastrepha obliqua (Macquart). High sterility values (> 98.2%) for irradiated males were obtained for doses as low as 25 Gy. Egg hatch was inhibited for irradiated males crossed with irradiated females at a low dose of 20 Gy. However, we estimated that to achieve 99.9% sterility (standard goal of many sterile insect technique programs), irradiation doses had to be increased to a dose between 50 and 75 Gy. At doses of 25 Gy and greater, we observed a decreasing trend in adult flight ability and an increasing trend in adult mortality. Such differences were greater for pupae irradiated at a young age compared those irradiated 24 h before emergence. Our single most relevant finding was that sterility induction (i.e., oviposition of nonfertilized eggs) was two times greater for males irradiated at low doses (40 Gy) than for males irradiated at high doses (80 Gy) when used at a 3:1:1 sterilized male to fertile male to fertile female ratio. Males irradiated at high doses may have been outcompeted by unirradiated males when courting unirradiated females. Implications of our findings for sterile insect technique programs are discussed.     

Competitive strategies between Anastrepha fraterculus and Anastrepha obliqua (Diptera: Tephritidae) regulating the use of host fruits

Interspecific competition between individuals of different species can result in reductions in their fecundity, growth or survival, reflecting differential exploitation of resources that become intensified due to spatial co-occurrence, ecological similarity and increased population densities. As two species cannot occupy the same niche, coexistence is only possible if the available resources are used in non-overlapping manners such as niche partitioning or the use of refuges. Among agricultural insect pests, such as fruit flies of the family Tephritidae, competitive interactions can result in competitive displacement, host changes, or the expansion or restriction of the numbers of hosts utilized that can have negative consequences for human agricultural activities. We evaluated the competitive interactions between two fruit fly species of the genus Anastrepha, Anastrepha obliqua (Macquart, 1835) and Anastrepha fraterculus (Wiedmann, 1830), on their respective preferred hosts (mangoes and guava). Experiments of larval competition and competition for ovipositioning sites by adult females were performed to compare the parameters of larval development time, numbers of pupae and emerged adults and numbers of ovipositions in the presence or absence of interspecific competition. We observed that the interactions between those species were asymmetrical and hierarchical, and our results suggest a competitive displacement of A. fraterculus by A. obliqua when those two species are present on the same fruit, whether mangoes or guavas.     

Dietary self-selection and discrimination threshold in wild Anastrepha obliqua females (Diptera,Tephritidae)

Experiments were performed on adult wild Anastrepha obliqua females to (I) determine whether there is a critical period during the reproductive phase when the absence of protein in the diet impairs egg production; (II) determine whether the females are able to self-select an optimal diet; and (III) determine the discrimination threshold for protein in newly emerged females as well as in 10-day old females that had either been deprived of protein or not deprived. The results showed that protein ingestion is essential during the preovipositing phase to allow egg production, that females are self-selective and that this behavior changes with age according to the reproductive status, and that protein deprivation strongly alters the discrimination threshold for protein. Newly emerged females have discriminatory thresholds similar to protein-deprived insects.     

Olfactory response of Anastrepha striata (Diptera: Tephritidae) to guava and sweet orange volatiles

The behavioral responses of virgin and mated female Anastrepha striata Schiner (Diptera: Tephritidae) to guava (Psidium guajava L.) or sweet orange (Citrus sinensis L.) were evaluated separately using multilure traps in two‐choice tests in field cages. The results showed that flies were more attracted to guava and sweet orange volatiles than to control (unbaited trap). The physiological state (virgin or mated) of females did not affect their attraction to the fruit volatiles. Combined analysis of gas chromatography coupled with electroantennography (GC‐EAD) of volatile extracts of both fruits showed that 1 and 6 compounds from orange and guava, respectively elicited repeatable antennal responses from mated females. The EAD active compounds in guava volatile extracts were identified by gas chromatography‐mass spectrometry (GC‐MS) as ethyl butyrate, (Z)‐3‐hexenol, hexanol, ethyl hexanoate, hexyl acetate, and ethyl octanoate. Linalool was identified as the only antennal active compound in sweet orange extracts. In field cage tests, there were no significant differences between the number of mated flies captured by the traps baited with guava extracts and the number caught by traps baited with the 6‐component blend that was formulated according to the relative proportions in the guava extracts. Similar results occurred when synthetic linalool was evaluated against orange extracts. From a practical point of view, the compounds identified in this study could be used for monitoring A. striata populations.     

Nonhost status of commercial Persea americana 'Hass' to Anastrepha ludens, Anastrepha obliqua, Anastrepha serpentina, and Anastrepha striata (Diptera: Tephritidae) in Mexico

The objective of this study was to determine the host status in Mexico of commercially cultivated and marketed avocado, Persea americana (Mill.), 'Hass' to Anastrepha ludens (Loew), Anastrepha obliqua (Macquart), Anastrepha serpentina (Wiedemann), and Anastrepha striata (Schiner) (Diptera: Tephritidae). Experiments in Michoacán, Mexico, were carried out in six orchards located at three altitudes above sea level during two times (August-October 2001 and April-June 2002). They included choice ('Hass' avocado plus natural host) and no-choice foraging behavior tests on trees under field cages; no-choice, forced infestation trials on caged, fruit-bearing branches in the field, and with individual fruit under laboratory conditions; infestation trials using 'Hass' avocados left unprotected over 1 and 7 d on the ground of orchards; studies to ascertain depth of oviposition and determine egg hatchability; and experiments to determine susceptibility by using time elapsed since removal of fruit from tree as the experimental variable. We trapped adult Anastrepha (n = 7,936) in all orchards and dissected fruit (n = 7,695) from orchards and packing houses (n = 1,620) in search of eggs or larvae. Most (96.7%) A. ludens, A. obliqua, A. striata, and A. serpentina adults were captured in low-elevation orchards. No eggs or larvae were detected in any of the fruit from foraging behavior studies or dissected fruit from orchards or packing houses. Of 5,200 mature, intact fruit on trees in the field forcibly exposed to no-choice female oviposition activity (five females/fruit), we only found four fruit infested by A. ludens but no adults emerged. 'Hass' avocados only became marginally susceptible to attack by A. ludens (but not A. obliqua, A. serpentina, and A. striata) 24 h after being removed from the tree. Fruit placed on the ground in orchards (n = 3,600) were occasionally infested by Neosilba batesi (Curran) (Diptera: Lonchaeidae), a decomposer, but not Anastrepha spp. Based on our results, commercially cultivated and marketed P. americana 'Hass' should not be considered a natural host of A. ludens, A. obliqua, A. striata, and A. serpentina in Mexico.     

Mating time of the West Indian fruit fly Anastrepha obliqua (Macquart) (Diptera: Tephritidae) under laboratory conditions

Allochronic reproductive isolation seems to be an important factor in speciation processes in Tephritidae since specific mating times are a widespread feature of its species. The timing of matings of the West Indian fruit fly, Anastrepha obliqua (Macquart) was investigated through group-focal observations, during ten days, under laboratory conditions. The number of observed matings and males exhibiting calling behavior varied significantly according to time of day. Sexual activities seemed to be concentrated in the afternoon period, with the male calling behavior reaching a peak between 3:30p.m. and 4:30p.m., and mating occurred most frequently from 2:00 p.m. to 4:30 p.m.     

Wolbachia in Anastrepha Fruit Flies (Diptera: Tephritidae)

Endosymbiotic bacteria of the genus Wolbachia are widespread among arthropods and cause a variety of reproductive abnormalities, such as cytoplasmic incompatibility, thelytokous parthenogenesis, male-killing, and host feminization. In this study, we used three sets of Wolbachia-specific primers (16S rDNA, ftsZ, and wsp) in conjunction with the polymerase chain reaction (PCR), cloning and sequencing to study the infection of fruit flies (Anastrepha spp. and Ceratitis capitata) by Wolbachia. The flies were collected at several localities in Brazil and at Guayaquil, Ecuador. All of the fruit flies studied were infected with Wolbachia supergroup A, in agreement with the high prevalence of this group in South America. Phylogenetic analysis showed that the wsp gene was the most sensitive gene for studying the relationships among Wolbachia strains. The Wolbachia sequences detected in these fruit flies were similar to those such as wMel reported for other fruit flies. These results show that the infection of Anastrepha fruit flies by Wolbachia is much more widespread than previously thought.     

Sexual receptivity of Anastrepha obliqua (Diptera: Tephritidae) wild females affected by nutrition and egg load

The influence of feeding, egg load and stage of ovarian development on the sexual receptivity of some insect species has been reported. This work investigated the effects of different brewer's yeast concentrations in the diet offered to females of West Indian fruit fly, Anastrepha obliqua, on their sexual receptivity. Wild females were fed on artificial diets containing 2.0 g brewer's yeast per 100 mL of water (low‐yeast diet, LY diet), 6.5 g yeast per 100 mL of water (high‐yeast diet, HY diet) or only sucrose (Su diet) and then placed with the males. Female sexual receptivity tests were carried out in acrylic boxes in the laboratory under controlled conditions (temperature, humidity and light). Females fed on the HY diet were more sexually receptive to male courtship than the other females. Additionally, when fed on the same diet (HY diet), those females with higher egg load were more sexually receptive. The data suggest that the ingestion of a protein source is essential for increasing sexual receptivity and that egg load is the most important factor affecting sexual receptivity of A. obliqua females.     

Anastrepha ludens and Anastrepha serpentina (Diptera: Tephritidae) do not infest Psidium guajava (Myrtaceae), but Anastrepha obliqua occasionally shares this resource with Anastrepha striata in nature

This study examined whether economically important fruit fly species Anastrepha ludens (Loew), Anastrepha serpentina (Wiedemann), and Anastrepha obliqua (Macquart) (Diptera: Tephritidae) may opportunistically exploit guavas, Psidium guajava L. (Myrtaceae), growing near preferred natural hosts. We collected 3,459 kg of guavas and 895 kg of other known host species [sour orange, Citrus aurantium L.; grapefruit, Citrus paradisi Macfadyen; mango, Mangifera indica L.; white sapote, Casimiroa edulis La Llave and Lex.; sapote, Pouteria sapota (Jacq.); sapodilla, Manilkara zapota L.; and wild plum, Spondias purpurea L. and Spondias mombin L.] along an altitudinal gradient over a 4-yr period (2006-2009). Plants were growing in sympatry in 23 localities where the guavas are usually infested in the state of Veracruz, M6xico. The guava samples yielded 20,341 Anastrepha spp. pupae in total (overall mean, 5.88 pupae per kg of fruit). Confirming previous reports, Anastrepha fraterculus (Wiedemann) and Anastrepha striata (Schiner) were found heavily infesting guavas in Veracruz. Importantly, although we did not find evidence that A. ludens and A. serpentina are able to attack this valuable commodity, we document for the first time in the agriculturally important state of Veracruz that P. guajava is an alternative natural host plant of A. obliqua. We recovered two fruit in the mango-growing locality of la Vibora, Tlalixcoyan, that harbored larvae of A. striata and A. obliqua. This finding has important practical implications for management of A. obliqua. Over the entire altitudinal gradient, when individual fruit infestation was examined, a dynamic pattern of species dominance was unveiled with guavas growing below 800 m above sea level mainly attacked by A. striata and a progressive replacement with increasing altitude by A. fraterculus. Interestingly, most individual fruit examined (97%) harbored a single species of fruit fly, a finding that may be taken as evidence of competitive displacement among sympatric species of fruit flies. Based on this study and previously published work by us on this topic, we conclude that literature reports indicating that A. ludens and A. serpentina infest guavas under field conditions should be questioned.     

Abiotic factors affecting the infectivity of Steinernema carpocapsae (Rhabditida: Steinernematidae) on larvae of Anastrepha obliqua (Diptera: Tephritidae)

The effects of soil depth, soil type and temperature on the activity of the nematode Steinernema carpocapsae (Filipjev) were examined using larvae of the West Indian fruit fly, Anastrepha obliqua (Macquart). Bioassays involved applying infective juveniles (IJs) to the surface of sterilized sand in PVC tubes previously inoculated with fly larvae of two ages. The 50% lethal concentration (LC₅₀) values estimated for 6-day-old larvae were 9, 20 and 102 IJs/cm² in tubes containing 2, 5 and 8 cm depth of sand, respectively, whereas for 8-day-old larvae, LC₅₀ values were 16, 40 and 157 IJs/cm², respectively. The effect of soil texture on the activity of S. carpocapsae was tested by applying the corresponding LC₅₀ concentrations of nematodes to sand, sand–clay and loamy–sand soils. For 6-day-old larvae, soil type had a highly significant effect on infection with the highest percentages of infection observed in the sand–clay mixture (60–82% depending on depth) compared to 45–64% infection in sand and 23–30% infection in loamy–sand soil. A very similar pattern was observed in 8-day-old larvae except that infection rates were significantly lower than in younger larvae. There was a significant interaction between soil type and soil depth. The effect of three temperatures (19, 25 and 30°C) on infection was examined in sand–clay soil. The infectivity of S. carpocapsae was affected by temperature and soil depth and by the interaction of these two factors. Response surface analysis applied to second order multiple linear regression models indicated that the optimal temperature for infection of larvae of both ages was ~26°C, at a depth of 7.9 cm for 6-day-old larvae and <2 cm for 8-day-old larvae, resulting in a predicted 91.4% infection of 6-day-old larvae and 61.2% infection of 8-day-old larvae. These results suggest that S. carpocapsae may have the potential to control fruit fly pests in tropical ecosystems with warm temperatures and high soil moisture levels, although this assertion requires field testing.     

New mariner elements in Anastrepha species (Diptera: Tephritidae)

Mariner-like elements (MLE) are members from class II of transposable elements also known as DNA transposons. These elements have a wide distribution among different groups of organisms, including insects, which can be explained by horizontal and vertical gene-transfer. MLE families have been described in tephritid flies and other genera. During screening for Wolbachia bacteria in fruit flies of the genus Anastrepha, we discovered two sequences related to mariner-like elements. Based on these sequences, we designed primers that allowed us to isolate and characterize two new mariner-like elements (Anmar1 and Anmar2) in Anastrepha flies. These elements, which belong to the mellifera and rosa subfamilies have a low nucleotide diversity, and are probably inactive and acquired by vertical transfer. This is the first report of mariner-like transposons in flies found in South America.     

Consumption of sugars by Anastrepha suspensa (Diptera: Tephritidae)

We were interested in determining the feeding response of the Caribbean fruit fly, Anastrepha suspensa Loew (Diptera: Tephritidae), to various sugar concentrations to develop an improved bait for adults. We compared the consumption of 0.01-1.00 M concentrations of glucose, fructose, raffinose, and sucrose in no-choice tests for 24-h- and 6-d-old male and female flies. Sucrose was the most consumed sugar or within the most consumed group of sugars at 0.02-0.20 M concentrations. There were no differences in consumption among sugars at 0.01, 0.40, and 1.00 M. Consumption generally increased with increasing sugar concentration except that sucrose consumption peaked at 0.20 M. Twenty-four-hour females consumed less fructose than other sugars; 24-h males consumed more sucrose than fructose or raffinose, with an intermediate response to glucose. Females in the 6-d group consumed more sucrose than the other three sugars, whereas 6-d males exhibited no difference in consumption among sugars. In choice tests, flies consumed more sugar solution than water, but the difference between 0.20 M fructose and water was not significant for 24-h males or 24-h females. In choice tests between 0.20 M fructose and 0.20 M sucrose, both 24-h and 6-d females showed a preference for fructose. Males of both age classes showed no preference. These results indicate that the responses of flies to different sugars can vary by sugar, gender, and age.     

Effects of age, diet, female density, and the host resource on egg load in Anastrepha ludens and Anastrepha obliqua (Diptera: Tephritidae)

Oocyte counts, used as a measure of egg load, were compared among three different age groups (15, 30 and 45 days) of two polyphagous species of tephritid fruit flies, Anastrepha ludens and Anastrepha obliqua, which were exposed to varying conditions of diet (sucrose vs sucrose and protein), availability of oviposition substrate (present vs absent), adult female density (1, 2 and 4 females/cage), and semiochemical context (presence vs absence of male pheromones and fruit volatiles). In both species, oocyte counts were higher in older females and for females fed sucrose and protein than for females fed sucrose only. The presence of artificial oviposition substrates influenced oocyte counts in A. obliqua, but not in A. ludens. Female density influenced oocyte counts in both species. Females maintained in groups had higher egg loads than isolated females. Finally, preliminary evidence suggests that semiochemical context influenced oocyte counts. Counts were highest for females in a room containing both fruit volatiles and male pheromone, lowest for females in a room containing neither volatiles nor pheromone, and intermediate for females in rooms containing either volatiles or pheromone but not both. Our results suggest that egg load is influenced by environmental factors in different ways in these two species. Egg load in A. obliqua, a species whose host fruits are highly ephemeral, is responsive to access to the host resource. By contrast, in A. ludens, a species infesting less ephemeral fruit, female density and age played a more important role than host stimuli. The role of ovarian maturation and oviposition in mediating these effects, as well as implications for mass rearing and pest management, are discussed.     

A new blend of white sapote fruit volatiles as potential attractant to Anastrepha ludens (Diptera: Tephritidae)

The behavioral and electrophysiological responses of nonirradiated male and female Anastrepha ludens (Loew) (Diptera: Tephritidae), to white sapote, Casimiroa edulis Oerst. (Rutaceae), volatiles were investigated. Females flew upwind and landed more often on fruit than on artificial fruit in wind tunnel bioassays. Males flew upwind (but not landed) more frequently on fruit than on artificial fruit. Porapak Q volatile extracts of white sapote also elicited upwind flight and landing on artificial fruit for both sexes. Gas chromatography-electroantennographic detection analysis of white sapote extracts revealed that antennae of both sexes responded to eight compounds. Two peaks were unidentified because they did not separate from the solvent. Subsequent peaks were identified by gas chromatography-mass spectrometry as styrene, myrcene, 1,2,4-trimethylbenzene, 1,8-cineole, and linalool in a proportion of 50: 21: 0.5: 27: 1.5, respectively. Eight peaks were tentatively identified as beta-trans-ocimene. The number of A. ludens captured in multilure traps baited with the synthetic white sapote blend was higher than the flies captured by the multilure unbaited traps (control) in field cages. However, the number of flies captured by traps baited with the white sapote blend was not different from that of flies captured by traps baited with hydrolyzed protein. Using standard chemical ecology techniques, we found potential attractants from wild sapote fruit for monitoring and management of A. ludens population.     

Patterns of inner chorion structure in Anastrepha (Diptera: Tephritidae) eggs

The inner chorion structure of Anastrepha eggs from 16 species of various infrageneric taxonomic groups is described by scanning and transmission electron microscopy. The layers of the chorion, the outer egg membrane, are structurally similar. Furthermore, an additional trabecular layer (ATL) that exists in some species, together with other characteristics, facilitates the recognition of four patterns of chorion structuring: Pattern I, in which the ATL layer is absent, is found in Anastrepha amita, the Anastrepha fraterculus complex, Anastrepha obliqua, Anastrepha sororcula, Anastrepha suspensa and Anastrepha zenildae (fraterculus group), and Anastrepha bistrigata and Anastrepha striata (striata group); Pattern II in Anastrepha serpentina (serpentina group), Anastrepha grandis (grandis group) and Anastrepha pseudoparallela (pseudoparallela group), in which the ATL presents large open spaces with pillars; Pattern III, found in Anastrepha consobrina (pseudoparallela group), in which the ATL is composed of round cavities; and Pattern IV, found in Anastrepha alveata and Anastrepha pickeli (spatulata group), where the large ATL cavities are reticulated. Comparatively, the chorion structure in Anastrepha eggs is more complex than in eggs of other fruit flies, e.g., Bactrocera, Rhagoletis and Ceratitis.     

Effect of fly density and holding period on quality parameters of pre-release chilled adults of Anastrepha obliqua (Diptera: Tephritidae)

Packing, shipping, holding and releasing methods of sterile tephritid fruit flies for the control of pest populations have recently received special attention because they are the final steps in the application of the sterile insect technique (SIT). In this study, we examined the effect of four holding densities, 0.524, 0.599, 0.674 and 0.748 sterile adults/cm², and four holding periods (5, 6, 7 and 8 days) in sterile West Indian fruit flies, Anastrepha obliqua (Diptera: Tephritidae). The key parameters of adult fliers (AF, also known as absolute fliers) and adult survival under stress were measured. We also compared two methods to determine the percentage of AF (called the tray and sample methods) after the holding period and the chilling process. Our results indicate that differences in holding densities and holding periods may not affect the percentage of AF and the survival of sterile adults if they are supplied with enough food and water, allowing the release of higher numbers of sexually mature sterile males. When estimating the percentage of AF, the evaluated methods resulted equivalent and reliable, but the sample method showed a clear advantage by allowing the use of lower numbers of sterile flies to perform the test, which may represent important savings in SIT programmes. These findings will require further studies on sexual competitiveness in field cages to better determine the optimal management of sterile flies in FERFs.     

Calling behavior of mass-reared and wild Anastrepha serpentina (Diptera: Tephritidae)

The calling behavior of mass-reared and wild males of Anastrepha serpentina (Wiedemann) (Diptera: Tephritidae) was studied both in the laboratory and in field cage tests. In the laboratory, density (1, 5, and 10 males per container), age, and hour of day significantly affected calling behavior. Mass-reared males called independently of density, whereas wild males only called at densities of 5 and 10 individuals. Males of both strains started calling when they were 5-7 d old. The daily pattern of male calling was similar in both strains, starting at 0730 hours, and reaching a peak at 1330-1630 hours. Field cage tests showed that mass-reared males started calling when they were 5d old; the period of peak calling was when males were 8-9 d old. In contrast, wild males began calling when they were 10 d old, reaching peaks when males were 13, 15, and 18 d old. Wild males tended to form leks to call during each day of the experiment, whereas mass-reared males only formed leks during 2 d, both strains displaying very low levels. During field cage tests, males, independently of strain, displayed two calling peaks, one peak in the morning and one peak in the afternoon, whereas males observed in the laboratory only showed a single calling peak. The results are discussed in view of the effects of mass rearing A. serpentina males in relation to potential use of the sterile insect technique.     

Irradiation of Anastrepha ludens (Diptera: Tephritidae) revisited: optimizing sterility induction

Irradiation doses currently applied to sterilize Mexican fruit flies, Anastrepha ludens (Loew) (Diptera: Tephritidae), for release under the sterile insect technique eradication campaign in Mexico, were reviewed in an effort to increase sterile male performance in the field. A dose maximizing sterility induction into wild populations was sought by balancing somatic fitness with genetic sterility. Doses of 40, 60, and 80 Gy induced 95% or more sterility in all males, which in turn induced similar degrees of sterility into a cohort of wild flies in the laboratory. However, a low dose of 40 Gy was sufficient to completely suppress egg production in females. Similarly, a mild carryover of genetic damage might have been transferred to the F1 progeny of males irradiated at 40 Gy crossed with fertile wild females. Our results suggest that the 80-Gy dose currently applied in Mexico can be lowered substantially without jeopardizing program goals. This view could be strengthened by comparing performance of males irradiated at different doses under more natural settings. In general, we discuss the value of determining irradiation doses for pest species where females are more radiosensitive than males, by selecting the dose that causes 100% sterility in females.     

A transgenic embryonic sexing system for Anastrepha suspensa (Diptera: Tephritidae)

The Sterile Insect Technique (SIT) is a highly successful biologically-based strategy to control pest insect populations that relies on the large-scale release of sterilized males to render females in the field non-reproductive. For medfly, a mutant-based sexing system is available as well as a transgenic system where a tetracycline-suppressible (Tet-off) toxic molecule is female-specifically produced. However, the former classical genetic system took many years to refine, and the latter system results in female death by a poorly understood mechanism, primarily in the pupal stage after rearing costs have been incurred. Here we describe a Tet-off transgenic embryonic sexing system (TESS) for Anastrepha suspensa that uses a driver construct having the promoter from the embryo-specific A. suspensa serendipity α gene, linked to the Tet-transactivator. This was used to drive the expression of a phospho-mutated variant of the pro-apoptotic cell death gene, Alhid, from Anastrepha ludens. The system uses a sex-specific intron splicing cassette linked to a cell death gene lethal effector. Progeny from TESS strains heterozygous for the transgene combination were 80–100% males, whereas four double homozygous TESS strains had 100% male-only progeny, with female death limited primarily to embryogenesis. In a large-scale test, more than 30,000 eggs from two strains resulted in 100% male-only progeny. The transgenic sexing approach described here is highly effective and cost-efficient by eliminating most, if not all, female insects early in embryogenesis using a well-characterized apoptotic mechanism.