Methoprene application and diet protein supplementation to male melon fly,Bactrocera cucurbitae,modifies female remating behavior

Methoprene （an analogue of juvenile hormone） application and feeding on a protein diet is known to enhance male melon fly, Bactrocera cucurbitae Coquillett （Diptera： Tephritidae）, mating success. In this study, we investigated the effect of these treatments on male B. cucurbitae＇s ability to inhibit female remating. While 14-d-old females were fed on protein diet, 6-d-old males were exposed to one of the following treatments： （i） topical application of methoprene and fed on a protein diet; （ii） no methoprene but fed on a protein diet; （iii） methoprene and sugar-fed only; and （iv） sugar-fed, 14-d-old males acted as controls. Treatments had no effect on a male＇s ability to depress the female remating receptivity in comparison to the control. Females mated with protein-deprived males showed higher remating receptivity than females first mated with protein-fed males. Methoprene and protein diet interaction had a positive effect on male mating success during the first and second mating of females. Significantly more females first mated with sugar-fed males remated with protein-fed males and females first mated with methoprene treated and protein-fed males were more likely to remate with similarly treated males. Females mating latency （time to start mating） was significantly shorter with protein-fed males, and mating duration was significantly longer with protein-fed males compared with protein-deprived males. These results are discussed in the context of methoprene and/or dietary protein as prerelease treatment of sterile males in area-wide control of melon fly integrating the sterile insect technique （SIT）.     

Methoprene application and diet protein supplementation to male melon fly,Bactrocera cucurbitae,modifies female remating behavior

Methoprene (an analogue of juvenile hormone) application and feeding on a protein diet is known to enhance male melon fly, Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), mating success. In this study, we investigated the effect of these treatments on male B. cucurbitae's ability to inhibit female remating. While 14‐d‐old females were fed on protein diet, 6‐d‐old males were exposed to one of the following treatments: (i) topical application of methoprene and fed on a protein diet; (ii) no methoprene but fed on a protein diet; (iii) methoprene and sugar‐fed only; and (iv) sugar‐fed, 14‐d‐old males acted as controls. Treatments had no effect on a male's ability to depress the female remating receptivity in comparison to the control. Females mated with protein‐deprived males showed higher remating receptivity than females first mated with protein‐fed males. Methoprene and protein diet interaction had a positive effect on male mating success during the first and second mating of females. Significantly more females first mated with sugar‐fed males remated with protein‐fed males and females first mated with methoprene treated and protein‐fed males were more likely to remate with similarly treated males. Females mating latency (time to start mating) was significantly shorter with protein‐fed males, and mating duration was significantly longer with protein‐fed males compared with protein‐deprived males. These results are discussed in the context of methoprene and/or dietary protein as prerelease treatment of sterile males in area‐wide control of melon fly integrating the sterile insect technique (SIT).     

Effect of conditions in sealed plastic bags on eclosion of mass‐reared Queensland fruit fly,Bactrocera tryoni

The sterile insect technique has been used for more than 50 years to control a range of insects around the world. Sterile insect technique is rapidly becoming a major component of many area‐wide fruit fly management programmes. Irradiation of immature life stages induces sterility in adults, which are then distributed over large areas to mate with wild flies, resulting in no viable offspring. However, irradiation in normal air results in declining adult quality. To optimize the quality of sterile adult flies, several techniques are available to lower the levels of oxygen in fruit fly tissues prior to irradiation. The simplest method is to seal pupae in plastic bags and allow the oxygen consumption of pupae to minimize oxygen in both the air and pupal tissue. Some fruit fly species have rapid decreases in eclosion as a result of low oxygen atmospheres. We tested the tolerance of Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), to low oxygen for the first time. In the first two experiments, unirradiated B. tryoni pupae were tested for different periods in sealed plastic bags at 17, 21, and 26 °C. Optimum eclosion occurred at 21 °C with the lowest eclosion at 26 °C. In general, mean full eclosion declined at ca. 0.1% eclosion per hour sealed in plastic bags during the first 96 h for all temperatures. In the third and fourth experiments at 17 °C, there was a decline in average eclosion for irradiated and unirradiated pupae of about 13.4% after they were sealed in plastic bags for 192 h. In general, B. tryoni eclosion declined at 0.1% per hour inside sealed plastic bags for periods up to 192 h at 17 °C. Queensland fruit flies can tolerate long periods of conditions found inside sealed plastic bags and current practices for sterile B. tryoni release programmes will result in minimum decrease in eclosion. The possible evolution of tolerance of these conditions is discussed.     

Rapid evolution of larval development time during mass-rearing in the melon fly, Bactrocera cucurbitae

A quantitative genetic analysis of rapid evolution of a life history trait has been conducted on the first 24 generations of mass-rearing in the melon fly Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). The phenotypic variance of larval development time in each generation was divided into genetic and residual components. Mean and phenotypic coefficients of variation of larval development time decreased gradually as generations proceeded as a result of artificial selection for shorter larval period in the mass-rearing procedure. There was a trend that additive genetic coefficients of variation in larval development time decreased with generations. These changes are entirely attributed to genetic responses to laboratory selection under the mass-rearing environment because the population was maintained at a very large size so as to exclude random genetic drift and inbreeding depression, which would be other factors responsible for the observed genetic changes. The residual coefficients of variation in larval development time did not change with generations. Realized heritability of larval development time was low. The heritabilities for larval development time estimated from parent–offspring regression at generations 60 and 70, when the evolutionary plateau was asymptotically reached, were not significantly larger than 0. Received: April 22, 1999 / Accepted: September 20, 1999     

Fecundity,fertility and reproductive recovery of irradiated Queensland fruit fly Bactrocera tryoni

Pupae of the Queensland fruit fly or Q‐fly Bactrocera tryoni (Froggatt) are irradiated routinely to induce reproductive sterility in adults for use in sterile insect technique programmes. Previous studies suggest that adult sexual performance and survival under nutritional and crowding stress are compromised by the current target dose of radiation for sterilization (70–75 Gy), and that improved mating propensity and survival under stress by irradiated males may be achieved by reducing the target sterilization dose without reducing the level of induced sterility. This raises the question of the amount by which the irradiation dose can be reduced before residual fertility becomes unacceptable. The present study measures the levels of residual fertility in male and female irradiated Q‐flies at different irradiation doses (20, 30, 40, 50, 60 and 70 Gy), and investigates the possibility that fecundity and fertility increase between 10–15 and 30–35 days post emergence. Male flies require a higher dose than females to induce sterility, with no residual fertility found in females irradiated at doses of 50 Gy or above, and no residual fertility found in males irradiated at doses of 60 Gy or above. Irradiated females are more fecund at 30–35 days post emergence than at 10–15 days. However, fertility does not increase between 10 and 15 days post emergence and 30–35 days, even at doses below 50 Gy. The present study shows that there is scope to reduce the target sterilization dose for Q‐flies below that of the current dose range (70–75 Gy) at the same time as retaining an adequate safety margin above radiation doses at which residual fertility can be expected.     

Response of female melon fly, Bactrocera cucurbitae, to host-associated visual and olfactory stimuli

In a series of studies conducted in Hawaii under seminatural conditions, we quantified the response of sexually mature, host‐seeking female melon flies, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae), to different types of visual and chemical host‐associated stimuli with the main aim of developing a monitoring device for females. Experiments were conducted using Tangletrap‐coated fruit mimics of either spherical (8 cm diameter) or cylindrical (4.3 cm diameter; 15 cm length) shapes coated with different artificial color pigments both at the ground level and at the tree‐canopy level so as to take into account the foraging behavior of adult melon flies. Females were particularly attracted to objects of spherical shape colored either yellow, white, or orange; these three pigments offered the highest reflectance values. Cucumber (Cucumis sativus L.) (Cucurbitaceae) odor was more attractive to females than odors of three other cultivated host fruit [zucchini, Cucurbita pepo L. var. medullosa Alef. (Cucurbitaceae); papaya, Carica papaya L. (Caricaceae); or tomato Solanum lycopersicum L. (Solanaceae)] or of ivy gourd [Coccinia grandis (L.) Voigt (Cucurbitaceae)], one of the major wild hosts of melon fly in Hawaii. A combination of both visual and olfactory stimuli was needed to elicit high levels of response compared to each stimulus offered alone. We discuss our results in relation to the potential implementation of improved female monitoring and/or attract‐and‐kill strategies for melon flies in Hawaii.     

Effects of field cage colour and supplementary shade on environmental conditions and mating behaviour of Queensland fruit flies,Bactrocera tryoni

The mating performance field cage test is a required periodic quality‐control assessment for factory‐reared fruit flies used for the sterile insect technique. The FAO/IAEA/USDA guidelines for assessing fly quality state that if during tests a large proportion of flies call and mate on cage walls, away from host trees, then environmental conditions within the cage need to be adjusted and tests repeated. Here we test effects of cage design, specifically mesh colour (green, white) and addition of supplementary shade, on the mating behaviour of Queensland fruit fly (Q‐fly), Bactrocera tryoni Froggatt (Diptera: Tephritidae). Observations were made over a 4‐h period at dusk when these flies mate. Changes in environmental conditions in each cage over the dusk period varied with cage design. We recorded the highest proportion of matings taking place on trees as opposed to cage walls (>90%) in the unshaded white cage, the shaded white and un‐shaded green cages being intermediate (ca. 70%), and the shaded green cage had the least (ca. 40%). The effects of field cage colour and supplementary shade on mating behaviour are discussed. We recommend that Q‐fly field cage tests should be conducted in cages with a light coloured mesh, and that supplementary shading should only be applied if there is a need to adjust temperature and light within the cage.     

Effect of diet on survival,in the laboratory and the field,of sterile male Mediterranean fruit flies

To improve the effectiveness of the sterile insect technique against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), our objectives in this study were two‐fold. First, to evaluate the ability of sterile males of the Vienna‐8 strain to survive starvation, we compared them to wild males under laboratory conditions. The second objective was to determine the effect of protein‐rich nutrition on sterile male fly survival, under starvation conditions in the laboratory, under semi‐natural conditions in a field enclosure, and under natural conditions in the open field. Therefore, we released marked sterile flies of the two diet regimes, protein‐fed or protein‐deprived, and monitored their survival by recapturing them after 4, 6, and 7 days. In the laboratory, wild males endured starvation significantly better than sterile ones and protein addition to sterile fly diet resulted in even greater reduced capability to endure starvation. On the other hand, the addition of protein to sterile‐male diet did not affect their ability to survive in a field enclosure or in the open field. We conclude that under natural conditions, where food is available, sterile male fly survival is unaffected by protein‐rich pre‐release diet.     

Effects of post-teneral diet on foraging success of sterile male Mediterranean fruit flies

Post‐teneral diets containing protein have been shown to enhance the copulatory success of sterile male Mediterranean fruit flies, Ceratitis capitata (Wied.) (Diptera: Tephritidae). However, ingesting protein was also found to negatively affect male survival, in particular when males faced starvation following release in the field. Accordingly, the objective of the present study was to determine the effects of various post‐teneral diets, presented to sterile males prior to release, on their subsequent ability to forage for carbohydrates and protein in the field. Using mark‐release recapture and analytic biochemical methods, we found that both protein‐fed and protein‐deprived males foraged successfully for protein and sugar in a field enclosure when these resources were available. We conclude that protein‐fed sterile males are able to exploit sources of nutrition in the release environment, and their inability to overcome starvation is not a concern for control operations using the sterile insect technique.     

Aromatherapy on a large scale: exposing entire adult holding rooms to ginger root oil increases the mating competitiveness of sterile males of the Mediterranean fruit fly in field cage trials

The sterile insect technique (SIT) is widely used in integrated programs against fruit fly pests, particularly the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Unfortunately, the mass-rearing procedures inherent to the SIT often lead to a reduction in male mating competitiveness. One potential solution involves the pre-release exposure of males to specific attractants. In particular, male exposure to ginger root oil [Zingiber officinale Roscoe (Zingiberaceae); hereafter GRO] has been shown to increase mating success dramatically in field cage trials. Initial studies exposed small groups of males (25 individuals), but more recent work has demonstrated that GRO exposure involving standard storage boxes (containing ≈ 36 000 males) also results in enhanced mating performance. The objective of the present study was to determine whether aromatization of entire trailers, holding ≈ 14 million sterile males from a genetic sexing [temperature sensitive lethal (tsl)] strain, increases male mating success. Independent of the total dose, spatial distribution, or type of dispenser used, sterile males exposed to GRO for a 24-h period displayed greater mating success than non-exposed males in mating cage trials (in which tsl males competed against males from a standard, bisexual strain for females from this same standard strain). Averaged over all experiments, tsl males exposed to GRO obtained 54% of all matings compared to 38% for non-exposed tsl males, an increase of 42%. The implications of these findings for SIT programs against C. capitata are discussed.     

Enhancing mating performance after juvenile hormone treatment in Anastrepha fraterculus: a differential response in males and females acts as a physiological sexing system

Methoprene (a mimic of juvenile hormone) treatment can reduce the time required for sexual maturation in Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) males under laboratory conditions, supporting its use as a treatment for sterile males within the context of the sterile insect technique (SIT). We evaluated sexual behaviour, mating competitiveness of methoprene-treated males, and female readiness to mate after methoprene-treatment in field cages. The study involved two strains of A. fraterculus from Argentina and Peru, which show several polymorphisms in relation to their sexual behaviour. We also analyzed whether methoprene treatment affected male and/or female behaviour in the same way in these two strains. Methoprene-treated males were equally competitive with untreated mature males, and became sexually competitive 6 days after emergence (3–4 days earlier than untreated males). In contrast, methoprene did not induce sexual maturation in females or, at least, it did not induce a higher rate of mating in 7-day-old females. These results were observed both for the Argentina and the Peru strains. Altogether, our results indicate that methoprene treatment produces sexually competitive males in field cages. In the absence of a genetic sexing system, and when sterile males and females of A. fraterculus are released simultaneously, the fact that females do not respond as do males to the methoprene treatment acts as a physiological sexing effect. Therefore, in the presence of mainly sexually immature sterile females, released sexually mature sterile males would have to disperse in search of wild fertile females, thereby greatly reducing matings among the released sterile insects and thus enhancing sterile insect technique efficiency.     

Fractionated irradiation improves the mating performance of the West Indian sweet potato weevil Euscepes postfasciatus

• 1 The sterile insect technique (SIT) is widely used to suppress or eradicate target pest insect populations.
• 2 The effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females. The use of gamma radiation to induce sterility, however, negatively affects both somatic cells as well as reproductive cells. Consequently, mating performance of sterilized individuals decreases drastically over time. The mating propensity of sterilized Euscepes postfasciatus (Fairmaire) males irradiated with a single dose of 150 Gy (the current standard of the Okinawa Prefecture SIT programme) is equal to that of non‐irradiated weevils for the first 6 days.
• 3 Fractionated irradiation, in which a sterilizing dose is delivered over time in a series of smaller irradiations, reduces the damage of irradiation in insects. In the present study, we evaluated the effect of fractionated irradiation on male fertilization ability, longevity and mating propensity of E. postfasciatus for a period of 16 days after irradiation.
• 4 Although fractionated irradiation totalling 150 Gy was found to induce full sterility regardless of the number of individual doses, the mating propensity of male weevils sterilized by fractionated irradiation was maintained for the first 12 days. These results demonstrate that fractionated irradiation can be highly advantageous in programmes aimed at eradication of E. postfasciatus.

     

A review of 16 years of quality control parameters at a mass‐rearing facility producing Queensland fruit fly,Bactrocera tryoni

From 1996 to 2012, the mass‐rearing facility at Camden (NSW, Australia) has been producing Queensland fruit flies, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae). During this time, the facility has regularly recorded fly quality parameters, creating a unique data set that provides an invaluable opportunity to evaluate the interrelationships among standard quality control (QC) parameters and test for redundant QC variables. Here, we conducted an exploratory data analysis to reveal relationships among the QC parameters. We found that pupal weight, adult lifespan, and longevity under nutritional stress (i.e., survival duration without food or water) had distinct monthly trends, suggesting that these QC parameters are sensitive to seasonal conditions. Furthermore, emergence percentage, flight ability, and adult lifespan were adversely affected by the dyeing/handling/irradiation process associated with sterile insect releases. Using a multivariate approach and controlling for monthly and yearly patterns, we showed that pupal weight and egg hatch are consistently negatively related and that percentage male and emergence rates are consistently negatively related. These results suggest that these correlation pairs measure similar quality information and hence one QC variable from each pair could be dropped. Flight ability was not strongly correlated with any of the QC variables, suggesting that this QC variable remains a useful QC metric. Finally, the longevity under nutritional stress QC appears to be fairly insensitive to QCs and we suggest that it should be replaced by the standard mortality under stress test.     

Female mating status does not affect male mating behavior in the West Indian sweetpotato weevil, Euscepes postfasciatus

Because multiple mating by females encourages sperm competition, the assessment of female mating status before insemination is important for males in order to avoid the risk of sperm competition or to intensify sperm competition. When interacting with females before sperm transfer, males can alter their mating tactics according to the risk or intensity of sperm competition. Information on how mating systems are associated with sperm competition is essential for sterile insect technique eradication programs, which depend on successful mating of released sterile males with wild females. We tested whether males of the West Indian sweetpotato weevil, Euscepes postfasciatus (Fairmaire) (Coleoptera: Curculionidae), adjusted their mating behavior in response to female mating experience and/or age. As virgin/young female weevils accepted males more easily than non-virgin/old females, assessing females before insemination can be adaptive for male weevils. We found that E. postfasciatus males were unable to adjust their mating tactics in response to female mating status. Although this mating strategy would be costly for individual male weevils, the ability in males to discriminate female mating status can prevent the chance of mating with already-mated females containing the sperm of wild male(s). Therefore, the mating tactics of male E. postfasciatus are advantageous for sterile insect technique eradication programs.     

Evaluation of a pelleted diet for larval mass-rearing of Anastrepha ludens and Anastrepha obliqua

Most artificial diets used for mass-rearing of fruit fly larvae involve the purchase and independent weighing of each ingredient of the formulation, followed by mixing and diet preparation process. A ready-to-use formulation would avoid this time-consuming task and simplify the preparation procedure, leading to a more standardized diet. In this work, we compared life-history traits and survival and developmental parameters of Anastrepha ludens Loew and Anastrepha obliqua (Macquart) (Diptera: Tephritidae) reared on two formulations of an artificial diet. Two experiments were performed; first, a conventional non-pelleted diet was compared to a newly developed pelleted diet, which was mixed with water before being given to the larvae. The second experiment consisted of a comparison between the pelleted diet which was stirred in a mixer with water and dissolved preservatives and a pelleted diet which was only soaked in water with dissolved preservatives. We found that the pelleted diet allowed the mass-rearing of A. ludens and A. obliqua. Moreover, feeding larvae with pelleted diet led to increased weight of the larva by 7.4 and 11.8%, and of the pupa by 9.5 and 16.7% for A. ludens and A. obliqua, respectively, compared to conventional diet. For process evaluation, the number of activities, handling time, reception, storage, and larval diet preparation were recorded. The pelleted diet only needed soaking in water containing preservatives, thereby simplifying the mass-rearing process and saving time, space, and labour. Pelletizing is a versatile technology that can be adjusted for better physicochemical quality and reduced microbiological risk. This diet should be of particular interest for the mass-rearing process in ‘sterile insect technique’ applications.     

Impact of lek mating on the sterile insect technique: A modeling study

Modelling studies are presented which describe the effect of lek mating on the control of a wild population by sterile male release. The mixed leks are assumed to follow a Poisson-binomial distribution and the system includes three parts: territory defense, matings inside a lek and matings outside a lek. The effects of parameters on the hatchability are discussed. Among the parameters, sterile type effect (W_s), female choice (f_s) and mating competitiveness (C_m) are the most important. The application to determining the effects of sterile male release and on the proportion of sterile males required for eradication are also discussed.     

Sexual competitiveness of a transgenic sexing strain of the Mediterranean fruit fly, Ceratitis capitata

The sterile insect technique (SIT), when used for the control of the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), generally relies on the release of sterile flies of only the male sex. Male selection is achieved through the use of a genetic sexing strain (GSS) in which females are killed by heat treatment in the generation prior to release. Transgenic sexing strains (TSS) have been developed that perform the same function of female-lethality, this time by withholding tetracycline (or related compounds) from the larval diet. The use of TSS may allow for certain problems associated with conventional GSS, such as strain instability and reduced productivity in mass-rearing, to be avoided. The performance, and principally the sexual competitiveness, of released male flies is important for the success of an SIT control programme. This study describes field cage experiments in which the competitiveness of males from a TSS (OX3376B) was compared with that of a conventional GSS (VIENNA-8) and two wild-type strains (TOLIMAN and ARG). When competing for female mates with wild-type males, OX3376B male performance was acceptable. When OX3376B males competed directly for mates with VIENNA-8 males, VIENNA-8 slightly outperformed the TSS males. Parallel tests, in which wild-type males competed with either OX3376B or VIENNA-8 males, showed that males from both sexing strains were highly competitive with wild-type males. These results suggest that OX3376B in particular, and TSS in general, show sufficiently good mating competitiveness to merit further research into their suitability for eventual use in SIT programmes.     

Evaluation of yeasts in gel larval diet for Queensland fruit fly,Bactrocera tryoni

Queensland fruit fly, Bactrocera tryoni (“Q‐fly”), is Australia’s most economically important insect pest of horticultural and commercial crops especially in the eastern regions. The sterile insect technique (SIT) has been adopted as an environmentally benign and sustainable approach for management of Q‐fly outbreaks. High‐performance larval diets are required to produce the millions of flies needed each week for SIT. Yeast products contribute amino acids (protein) to fruit fly larval diets, as well as carbohydrate, fat and micronutrients, but there can be substantial variation in the nutritional composition and suitability of yeast products for use in larval diets. Gel larval diets have recently been developed for large‐scale rearing of Q‐fly for SIT, and composition of these diets requires optimization for both performance and cost, including choice of yeast products. We assessed performance of Q‐flies reared on gel larval diets that contained debittered brewer’s yeast (Lallemand LBI2240), hydrolysed yeast (Lallemand FNILS65), inactivated brewer’s yeast (Lallemand LBI2250) and inactivated torula yeast (Lallemand 2160‐50), including blends. Q‐flies performed poorly when reared on diets containing only or mostly hydrolysed yeast in terms of pupal number, pupal weight and percentage of fliers. Performance was also poor on diets containing high proportions of torula yeast. Overall, debittered brewer’s yeast is recommended as the best option for Q‐fly gel larval diet, as it is cheap, readily available, and produces flies with good performance in quality control assays. Inactivated brewer’s yeast produced flies of comparable quality with only a modest increase in cost and would also serve as an effective alternative.     

Monitoring growth,survival and enzyme system of melon fruit fly Bactrocera cucurbitae (Coquillett) under the influence of affinity purified pea lectin

Pisum sativum agglutinin has been shown to act as a feeding inhibitor for various insect pests belonging to different orders: Lepidoptera, Coleoptera and Hemiptera. In the present study, its insecticidal activity was assessed through monitoring the growth and development of a dipteran pest Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Pea lectin, P. sativum agglutinin (PSA) was purified by single step affinity chromatography on a Sephadex G‐100 and the purification was monitored through hemagglutination activity and SDS‐PAGE. Insect feeding assays were conducted to determine the effect of pea lectin against first and second instar larvae of melon fruit fly B. cucurbitae. Lectin was incorporated in an artificial diet at a varied range of concentrations, 12.5, 25, 50, 100, 200 and 400 μg/mL. The lectin showed highly significant antimetabolic effects in both first and second instars. Time taken for pupation and development as well as percentage pupation and percentage adult emergence were adversely affected. The activity of three hydrolase enzymes (esterases, acid and alkaline phosphatases), five oxidoreductases (superoxide dismutase, catalase, ascorbate peroxidase, peroxidase, O‐demethylase) and one group transfer enzyme (glutathione‐S‐transferases) was also assessed in second instar larvae fed on lectin treated diet at 100 μg/mL concentration. The P. sativum lectin significantly and deleteriously influenced the activity of all these enzymes at all exposure intervals.