CRISPR/Cas9-mediated methoprene-tolerant 1 knockout results in precocious metamorphosis of beet armyworm (Spodoptera exigua) only at the late larval stage.

Juvenile hormone (JH) controls almost every aspect of an insect, especially metamorphosis. Since RNA interference works on transcripts and is often insufficient in Lepidoptera, how JH affects larval development in these insects is not well studied. Using the CRISPR/Cas9 technique, we knocked out Spodoptera exigua methoprene-tolerant 1 (SeMet1) gene of beet armyworm by modifying two sites in the coding region. However, SeMet1 knockout did not affect egg hatch rate or larval development at L1-L3 stages. In contrast to the consistent five larval instars of the control group, L4 SeMet1 mutants began to show signs of precocious metamorphosis, that is, small patches of pupal cuticle. Most L4 and all L5 SeMet1 mutants died for failing to shed their mosaic cuticles. RNA-seq indicated that most genes encoding pupal cuticle proteins and chitinase genes were altered in SeMet1 mutant L4 larvae. SeKr-h1, a key transcription factor in JH action was significantly down-regulated in L3-L5 larvae, while SeBR-C, a pupal indicator was only upregulated in L4-L5 larvae. These results suggested that S. exigua larvae may initially develop independently of JH, and involve SeMet1 in transducing JH signalling, leading to controlled larval metamorphosis at the late larval stage. We believe our findings will enhance better understanding of JH regulation of larval development.

Diallyl trisulfide reduced the reproductive capacity of male Sitotroga cerealella via the regulation of juvenile and ecdysone hormones.

Environmental pollution and resistance in animals are major concerns for the application of synthetic pesticides. Diallyl trisulfide (DAT), an active compound in garlic essential oil, is a novel tool for active and safe control of agricultural insect pests. In this study, we analysed the effects of DAT (0.01 µL/L) on the protein content in male reproductive tissues (accessory glands, ejaculatory ducts, and testis), and juvenile hormone (JH) and ecdysone titres in a highly detrimental pest of stored products, Sitotroga cerealella. Evaluation of the expression profile of JH and ecdysone pathway-related genes in various tissues indicated that the accessory gland protein and ecdysone titres were markedly decreased after DAT fumigation, whereas the testis protein content and JH titre were increased. However, the protein content of the ejaculatory ducts remained unchanged between the treated and control groups. Further investigation revealed that DAT disrupted the mRNA expression of key enzymes involved in JH and ecdysone pathways. While increased mRNA levels of juvenile hormone acid O-methyltransferase (JHMAT) and Kruppel homologue 1 (Kr-h1) were observed after 4 and 7 h of DAT fumigation, the levels of juvenile hormone epoxide hydrolase (JHEH) were substantially reduced 3 h post-fumigation. mRNA levels of the ecdysone-responsive gene, FTZF1, and cytochrome P450 enzyme, CYP315A1, were notably decreased at 7 h and 4 h, respectively, post-fumigation, whereas CYP314A1 and CYP302A1 mRNA levels decreased after 3 h and 4 h, respectively. While DAT fumigation disrupted sperm number in the testis, ejaculatory ducts, and seminal vesicles, topical application of the 20-hydroxyecdysone (20E) analogue also lowered sperm number in the ejaculatory ducts. Topical application of methoprene, a JH analogue, increased the protein content in the testes, but not in the accessory glands or ejaculatory ducts. However, the survival rate was not affected by the topical application of methoprene or 20E. These data suggest that DAT regulates JH and ecdysone via its molecular pathway genes and modulates endocrine secretion during the male reproductive process.

Pre-release dietary supplements of methoprene and raspberry ketone increase field abundance of sterile Queensland fruit flies (Diptera: Tephritidae).

The sterile insect technique (SIT) is a sustainable pest management tool based on the release of millions of sterile insects that suppress reproduction in targeted populations. Success of SIT depends on survival, maturation, dispersal, and mating of released sterile insects. Laboratory and field cage studies have demonstrated that dietary supplements of methoprene and raspberry ketone (RK) promote sexual maturation of adult Queensland fruit fly, Bactrocera tryoni (Froggatt), and may hence shorten the delay between release and maturity in the field. We investigated the effects of methoprene and RK dietary supplements on field abundance of sexually mature sterile Q-flies relative to untreated flies fed only sugar and yeast hydrolysate before release at 2 d of age. Compared with untreated flies, more methoprene- and RK-treated flies were recaptured in cuelure traps to which only sexually mature males are attracted. At distances of 100 and 200 m from the release point, recapture rates were higher for methoprene- and RK-treated flies than for untreated flies, but at 300 m recapture rates were low and were similar for treated and untreated flies. Rainfall, relative humidity, wind speed, and wind direction did not affect recapture rates, but temperature was positively correlated with recapture rates for all treatments. There was a strong correlation between the number of sterile and wild flies caught in traps, indicating co-location in the field. Dietary supplements of methoprene and RK can substantially increase abundance of sexually mature sterile male Q-flies in the field following release as 2-d-old immature adults.

Dietary methoprene treatment promotes rapid development of reproductive organs in male Queensland fruit fly.

Methoprene supplements added to diets of yeast hydrolysate and sugar promote early expression of sexual behaviour and mating in male Queensland fruit fly (Bactrocera tryoni; 'Q-fly') and show promise as a pre-release treatment for sterile insect technique programs. Currently it is not known whether the early mating behaviour of methoprene-treated male Q-flies is only behavioural or is coupled with accelerated development of reproductive organs. Accordingly, the present study investigates whether incorporation of methoprene into diets of yeast hydrolysate and sugar (1:3) or sugar alone, accelerate development of testes, ejaculatory apodeme, and accessory glands in male Q-flies and ovaries in females. All organs increased in size as the flies aged and matured, and development rate of all organs was far greater when the flies were provided yeast hydrolysate in addition to sugar. Incorporation of methoprene into diets containing yeast hydrolysate was found to strongly accelerate development of testes and ejaculatory apodeme, but not accessory glands, in males. In the absence of yeast hydrolysate, methoprene treatment had only a modest effect on male organ development. In contrast to males, development of ovaries in female Q-flies did not respond to dietary methoprene supplements, regardless of whether they were fed yeast hydrolysate and sugar or sugar alone. These findings of diet-dependent effects of methoprene supplements on reproductive organs are a close match to previous studies investigating effects of methoprene supplements on mating behaviour. Overall, methoprene supplements substantially enhance the positive effects of protein rich adult diet on the early expression of sexual behaviour and accelerate development of reproductive organs in male, but not female, Q-flies. Methoprene supplements added to pre-release diets of yeast hydrolysate and sugar show promise as a means of accelerating reproductive development of Q-flies released in sterile insect technique programs, and may also bias operational sex ratio in favour of males.

Combined effects of dietary yeast supplementation and methoprene treatment on sexual maturation of Queensland fruit fly.

Yeast hydrolysate supplements promote maturation of many tephritid flies targeted for control using the sterile insect technique (SIT), including Queensland fruit fly (Bactrocera tryoni; 'Q-fly'). Recently, application of the juvenile hormone analogue methoprene has been demonstrated to further promote maturation in some species. We here investigate the separate and combined effects of yeast hydrolysate and methoprene treatment on sexual maturation of sterile male and female Q-flies. Two methods of applying methoprene solution were used; topical application to adults and dipping of pupae. Consistent with previous studies, access to yeast hydrolysate greatly increased maturation of both male and female Q-flies. Maturation was further promoted by methoprene treatment, with similar effects evident for males and females and for both application methods. For flies provided access to yeast hydrolysate supplements, methoprene treatment advanced maturation by approximately 2days. No effects of diet or methoprene treatment were found on timing of copulation or copula duration. Countering the positive effects on sexual maturation, dipping of pupae in methoprene/acetone solution did diminish emergence rates and flight ability indices, and increased rates of wing deformity. Promising results of the present study encourage further investigation of treatment methods that maximise maturation while minimising detrimental effects on other aspects of fly quality.

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).

Precocious sexual signalling and mating in Anastrepha fraterculus (Diptera: Tephritidae) sterile males achieved through juvenile hormone treatment and protein supplements.

Sexual maturation of Anastrepha fraterculus is a long process. Methoprene (a mimic of juvenile hormone) considerably reduces the time for sexual maturation in males. However, in other Anastrepha species, this effect depends on protein intake at the adult stage. Here, we evaluated the mating competitiveness of sterile laboratory males and females that were treated with methoprene (either the pupal or adult stage) and were kept under different regimes of adult food, which varied in the protein source and the sugar:protein ratio. Experiments were carried out under semi-natural conditions, where laboratory flies competed over copulations with sexually mature wild flies. Sterile, methoprene-treated males that reached sexual maturity earlier (six days old), displayed the same lekking behaviour, attractiveness to females and mating competitiveness as mature wild males. This effect depended on protein intake. Diets containing sugar and hydrolyzed yeast allowed sterile males to compete with wild males (even at a low concentration of protein), while brewer´s yeast failed to do so even at a higher concentration. Sugar only fed males were unable to achieve significant numbers of copulations. Methoprene did not increase the readiness to mate of six-day-old sterile females. Long pre-copulatory periods create an additional cost to the management of fruit fly pests through the sterile insect technique (SIT). Our findings suggest that methoprene treatment will increase SIT effectiveness against A. fraterculus when coupled with a diet fortified with protein. Additionally, methoprene acts as a physiological sexing method, allowing the release of mature males and immature females and hence increasing SIT efficiency.

Efficacy of layer treatment with methoprene for control of Rhyzopertha dominica (Coleoptera: Bostrychidae) on wheat, rice and maize.

BACKGROUND: Insect growth regulators are promising alternatives to traditional pesticides in stored grain. The efficacy of the juvenile hormone analogue methoprene was evaluated as a layer treatment in a laboratory experiment for control of Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) in wheat, rice and maize. RESULTS: Adults of R. dominica were placed in vials containing 33, 26 and 29 g (to a depth of 6.5 cm) of wheat, rice and maize, respectively, that was entirely or partially treated with 1, 5 or 10 mg kg(-1) methoprene. In wheat and rice, the layer treatments were not as effective as the whole-grain treatment, but there was decreased progeny production as the application rate increased. However, on maize the partial treatments were as effective as the whole-grain treatment at 5 and 10 mg kg(-1) . CONCLUSIONS: The results suggest that partial layer treatments with methoprene can be used to control R. dominica on maize but may not be effective for control of this species on wheat and rice.

Morphologic effects of in vivo acute exposure to the pesticide methoprene on the hepatopancreas of a non-target organism, Homarus americanus.

Methoprene is a pesticide widely used for mosquito control. It is an endocrine disruptor, acting as an analog of juvenile hormone. While targeting insect larvae, it also impacts non-target animals including crustaceans. Anecdotal reports suggested that methoprene has unintended effects on adult arthropods. Earlier, we documented effects in adult lobsters at the metabolic and gene expression levels. In this study we have documented morphologic corollaries to our prior observations. We examined the light and electron microscopic changes in the hepatopancreas of adult lobsters following in vivo acute exposure to methoprene. Changes by light and electron microscopy levels were evident following exposure to sub-lethal concentrations of methoprene for 24h. Tissue from exposed animals showed the formation of extensive cytoplasmic spaces (vesiculation) with disruption and loss of specific subcellular organelles. The findings provide morphologic correlates to the metabolic and genomic alterations we have observed in previous investigations.

Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon fly Bactrocera cucurbitae: effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast.

The application of methoprene, and providing access to diet including hydrolyzed yeast, are treatments known to enhance mating success in the male melon fly Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), supporting their use in mass rearing protocols for sterile males in the context of sterile insect technique (SIT) programmes. The objective of the present laboratory study was to investigate the effect of methoprene application and diet supplementation with hydrolyzed yeast (protein) on the turnover of body lipids and protein to confirm the feasibility of their application in melon fly SIT mass-rearing programmes. While females had access to a diet that included hydrolyzed yeast (protein), males were exposed to one of the following treatments: (1) topical application of methoprene and access to diet including protein (M+P+); (2) only diet including protein (M-P+); (3) only methoprene (M+P-) and (4) untreated, only sugar-fed, control males (M-P-). Total body carbon (TBC) and total body nitrogen (TBN) of flies were measured at regular intervals from emergence to 35 days of age for each of the different treatments. Nitrogen assimilation and turnover in the flies were measured using stable isotope ((15)N) dilution techniques. Hydrolyzed yeast incorporation into the diet significantly increased male body weight, TBC and TBN as compared to sugar-fed males. Females had significantly higher body weight, TBC and TBN as compared to all males. TBC and TBN showed age-dependent changes, increasing until the age of sexual maturity and decreasing afterwards in both sexes. Methoprene treatment did not significantly affect TBC or TBN. The progressive increase with age of TBC suggests that lipogenesis occurs in adult male B. cucurbitae, as is the case in other tephritids. Stable isotope dilution was shown to be an effective method for determining N uptake in B. cucurbitae. This technique was used to show that sugar-fed males rely solely on larval N reserves and that the N uptake rate in males with access to diet including hydrolyzed yeast was higher shortly after emergence and then stabilized. The implications of the results for SIT applications are discussed.

Efficacy of the fipronil 10%+(S)-methoprene 9% combination against Rhipicephalus sanguineus in naturally infested dogs: speed of kill, persistent efficacy on immature and adult stages and effect of water.

This field trial was designed to test the efficacy, in terms of treatment and prevention, of the fipronil 10%+(S)-methoprene 9% combination against immature and adult stages of Rhipicephalus sanguineus in naturally infested dogs, and to assess the effect of a single plain water exposure. Twenty-four dogs of various age, sex, weight and coat length were divided into two homogeneous groups, treated (T) and control (C), and housed into twin outdoor kennels. Trial baseline was designed as day 0, when dogs from group T were treated with a commercial spot-on formulation of fipronil 10%+(S)-methoprene 9%, while subjects from group C were left untreated and served as control. After treatment, tick load for each included dog was estimated, for both adult and immature ticks, using the localization and count over the entire body surface at the following time-points: day 2, to evaluate the speed of kill and at days 7, 14, 21 and 28 to assess the persistence of efficacy. The effect of water exposure on the product efficacy was tested at day 14 of the study, when six dogs, homogenously selected from group T, were soaked through with plain water. The overall tick load in dogs from group C was high throughout the entire study period, ranging from 103.2 (day 28) to 161.3 (day 0), and confirmed the high tick pressure. Speed of kill calculated at 48 h post-treatment was slightly higher for adult ticks (96.2%) than for immature stages (91.6%). Compared to the control, dogs treated with the fipronil+(S)-methoprene maintained a significantly lower mean tick load for both adult and immature stages in the four weeks of observation. Persistence of efficacy against immature stages ranged from 97.1% the first week, 99.6% second week, 99.7 third week and 93.1% in the last week. In the same way, efficacy against adult ticks was constantly high, shifting from 94.5% to 92.5%. Overall efficacy (adults+immatures) was the strongest in the first two weeks (i.e., 96.1% and 96.6%) and lowered in the two remaining weeks (i.e., 94.2% and 93.4%). Results showed that water exposure did not affect the activity and persistence of the tested combination. On the basis of this field trial, the fipronil+(S)-methoprene combination represents a highly efficacious product to control all stages of R. sanguineus ticks on dogs.

Disruption of hepatic mitochondrial bioenergetics is not a primary mechanism for the toxicity of methoprene - relevance for toxicological assessment.

Methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) is an insect growth regulator generally used to control insect populations by preventing insect maturation. So far, the effects of the insecticide on mitochondrial bioenergetics were not investigated. In the present work, liver mitochondria from Wistar rats were isolated and features of mitochondrial physiology were characterized in the presence of methoprene. High concentrations of methoprene, in the range of 40-100 nmol/mg of protein could decrease the transmembrane electric potential (Delta Psi) developed by mitochondria and, at the highest concentration, methoprene prevented complete Delta Psi repolarization after ADP addition. The effect was more evident using succinate than with ascorbate+TMPD as substrate. State 3 respiration was approximately 60% inhibited by 80 nmol of methoprene/mg of protein, while state 4 respiration, within the same range of methoprene concentrations, showed a slight increase, when both glutamate-malate and succinate were used as substrates. Additionally, FCCP-stimulated respiration was inhibited to an extent comparable to the effect on state 3, which suggests an interaction of methoprene with the respiratory chain, more evident with glutamate/malate as substrate. The activity of complex I (NADH-ubiquinone oxidorreductase) and that of the segment comprehending complexes II and III (succinate-cytochrome c reductase) were decreased in the presence of methoprene (approximately 60% and 85% of inhibition, respectively, with 300 nmol of methoprene/mg of protein), while the activities of cytochrome c oxidase and ATPase do not seem to be affected. Furthermore, the action of methoprene on the mitochondrial permeability transition was also studied, showing that the insecticide (in the range of 30-80 nmol mg(-1) of protein) decreases the susceptibility of liver mitochondria to the opening of the transition pore, even in non-energized mitochondria. These results lead to the conclusion that methoprene interference with hepatic mitochondrial function occurs only for high concentrations, which implies that the noxious effects of the insecticide reported for a number of non-target organisms are not fully attributable to mitochondrial effects. Therefore, it seems that mitochondrial activity does not represent the primary target for methoprene toxic action.

Laboratory determination of efficacy of a Santalum spicatum extract for mosquito control.

The activity of QN50, a sequiterpene alcohol derived from Australian sandalwood (Santalum spicatum), was tested for its effectiveness against larvae of 2 mosquito species (Culex molestus and Aedes camptorhynchus [Diptera: Culicidael), nymphs of 2 species of water boatmen (Micronecta robusta and Agraptocorixa [Hemiptera: Corixidae]), immature Daphnia sp. (Crustacea), and mosquito eggs (Cx. molestus). In a series of laboratory bioassays, field-collected mosquito larvae, eggs, and immature corixids and daphnids were placed in beakers with either QN50, methoprene or source water only (control). The mosquito larvae exposed to QN50 had reduced survivorship and average longevity relative to the control and to methoprene at most concentrations used in this study. The hatching rate of mosquito eggs was unaffected by methoprene or QN50. Corixid nymphs and daphnids experienced high mortality in both methoprene and QN50 relative to the control, but there was no difference in the effect between the compounds. The results of this preliminary study suggest that further research into the mode of action and efficacy of QN50 as a potential alternative to methoprene for mosquito abatement is warranted.

Determination of altosid insect growth regulator in waters, soils, plants, and animals by gas-liquid chromatography.

Residues of isopropyl (2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate (Altosid) insect growth regulator are determined in waters, soils, plants, milk, eggs, fish, shellfish, poultry and cattle tissues, blood, urine, and feces. Acetonitrile is the primary extraction solvent for all samples. Residues are extracted by high-speed blending followed by vacuum filtration. Fatty extracts are subjected to cold-temperature precipitation and filtration. Samples are cleaned up by petroleum ether partitioning and Florisil and neutral alumina chromatography. The concentrated eluants are analyzed by gas-liquid chromatography (GLC) on columns of differing polarity, using hydrogen flame ionization detectors. The identity of suspected residues is confirmed by additional GLC and by mass fragmentography. The lower limits of detection were: water samples, 0.0004-0.001 ppm; soils, blood, and urine, 0.001 ppm; forage grasses, forage legumes, and rice foliage, 0.005 ppm; and milk, eggs, fish, shellfish, poultry and cattle tissues, and feces, 0.010 ppm.