Control of phlebotomine sandflies in confined spaces using diffusible repellents and insecticides

Abstract.  The control of phlebotomine sandflies (Diptera: Psychodidae), the vectors of leishmaniasis, is directed mostly against adults as larvae develop in unknown or inaccessible habitats. In the current study we tested geraniol, a natural plant-derived product, as a space repellent and the synthetic pyrethroid prallethrin as a diffusible insecticide. Geraniol was dispersed in the air using diffusers with an electric fan and prallethrin was evaporated using electrically heated evaporators. Both substances were tested in inhabited bedrooms and in tents. Geraniol failed to effect significant reductions in the numbers of either Phlebotomus papatasi Scopoli in rooms or Phlebotomus sergenti Parrot in tents. In laboratory experiments, geraniol proved ineffective in preventing sandflies from feeding. By contrast, prallethrin was highly effective in reducing the number of sandflies in rooms as well as in tents. Exposure of sandflies to prallethrin in laboratory experiments caused 97% mortality rates. Both prallethrin and, to a lesser extent, geraniol reduced the number of Culex mosquitoes captured in tents. Electric liquid-vaporizers with 1.5% prallethrin are highly effective in protecting people from sandfly bites in confined spaces and may be useful in combating cutaneous leishmaniasis.     

Natural breeding places of phlebotomine sandflies

Methods of finding larvae and pupae of phlebotomine sandflies (Diptera: Psychodidae) are described and the known types of breeding sites used by sandflies are listed. Three ways of detecting sandfly breeding places are the use of emergence traps placed over potential sources to catch newly emerged adult sandflies; flotation of larvae and pupae from soil, etc., and desiccation of media to drive out the larvae. Even so, remarkably little information is available on the ecology of the developmental stages of sandflies, despite their importance as vectors of Leishmania, Bartonella and phleboviruses affecting humans and other vertebrates in warmers parts of the world. Regarding the proven or suspected vectors of leishmaniases, information on breeding sites is available for only 15 out of 29 species of sandflies involved in the Old World and 12 out of 44 species of sandflies involved in the Americas, representing approximately 3% of the known species of Phlebotominae. Ecotopes occupied by immature phlebotomines are usually organically rich moist soils, such as the rain forest floor (Lutzomyia intermedia, Lu. umbratilis, Lu. whitmani in the Amazon; Lu. gomezi, Lu. panamensis, Lu. trapidoi in Panama), or contaminated soil of animal shelters (Lu. longipalpis s.l. in South America, Phlebotomus argentipes in India; P. chinensis in China; P. ariasi, P. perfiliewi, P. perniciosus in Europe). Developmental stages of some species (P. langeroni and P. martini in Africa; P. papatasi in Eurasia; Lu. longipalpis s.l. in South America), have been found in a wide range of ecotopes, and many species of sandflies employ rodent burrows as breeding sites, although the importance of this niche is unclear. Larvae of some phlebotomines have been found in what appear to be specialized niches such as Lu. ovallesi on buttress roots of trees in Panama; P. celiae in termite hills in Kenya; P. longipes and P. pedifer in caves and among rocks in East Africa. Old World species found as immatures in the earthen floor of human habitations include P. argentipes, P. chinensis, P. martini and P. papatasi. Much more information on sandfly breeding sites is required to facilitate their control by source reduction.     

Control of phlebotomine sandflies

Phlebotomine sandflies (Diptera: Psychodidae) transmit many zoonotic diseases (arboviruses, bartonelloses and especially leishmaniases) of importance to human health in at least 80 countries. Measures used to control adult sandflies (Lutzomyia and Phlebotomus) include the use of insecticides (mostly pyrethroids) for residual spraying of dwellings and animal shelters, space-spraying, insecticide-treated nets, impregnated dog-collars and personal protection through application of repellents/insecticides to skin or fabrics. Because the breeding-sites of sandflies are generally unknown, control measures that act specifically against immatures are not feasible, although the effectiveness of a few biological and chemical agents has been demonstrated in laboratory evaluations. Reports of insecticide-resistance refer to only three sandfly species (P. papatasi, P. argentipes and S. shorttii) against DDT in one country (India), although there are reports of DDT-tolerance in several countries. Current knowledge of sandfly susceptibility to various insecticides is summarized. Constraints and advantages of different compounds, formulations and delivery methods for sandfly control under different environmental conditions are discussed.     

Formulation of pyriproxyfen, a juvenile hormone mimic, for tsetse control

A topical dose, in 1 microliter acetone, of 0.02 microgram-2-[1-methyl-2-(4-phenoxyphenoxy) ethoxy] pyridine, the juvenile hormone mimic pyriproxyfen (S-31183, Sumitomo Chemical Co.), caused an adult female tsetse, Glossina morsitans morsitans Westwood, to produce non-viable offspring for the whole of her life. Using 14C labelled pyriproxyfen it was determined that as little as 0.001 microgram transferred to the in utero larva was sufficient to arrest development in the pupal stage. A formulation in vegetable oil was prepared for treating black cotton cloth targets which caused females to pick up 0.1 microgram active ingredient (a.i.) by tarsal contact during 1 min of exposure. Males exposed similarly for between 1 and 5 min transferred up to 0.016 microgram a.i. to females if they mated immediately after treatment. Doses as low as 0.01 micrograms in 10 microliters oil cm-2 on black cotton cloth targets caused females to produce non-viable offspring for at least two reproductive cycles following exposure. However, a dose of 0.1 microgram in 10 microliters oil cm-2 was necessary for an exposed male to cause disruption of the reproductive potential of his mate. This juvenile hormone mimic has potential to induce sterility via both sexes of tsetse using treated targets or traps under field conditions.     

保幼激素类似物对斜纹夜蛾核多角体病毒增殖的影响

分别用5×10⁶,1×10⁷,5×10⁷,1×10⁸ PIBs/mL 4种浓度的斜纹夜蛾核多角体病毒(SpltNPV)感染斜纹夜蛾末龄幼虫（第6龄）,并于同龄期以10μg/头的保幼激素类似物(JHA) methoprene分别对各感染组进行点滴处理,以研究JHA对SpltNPV增殖的影响。研究表明,与对照组相比各不同浓度处理组病毒总产量分别提高227.06%、128.71%、52.62%、33.15%,平均单头病毒含量分别提高49.15%、48.40%、36.40%和31.11%,病毒感染死亡率分别提高119.21%、52.72%、12.64%和1.12%。其中以1×10.7 PIBs/mL感染再经JHA处理的病毒总产量和平均单头病毒含量最高,分别为1 701.8×10⁸PIBs和60.1×10⁸ PIBs。各处理组的病毒总产量和平均单头病毒含量均显著高于其对照组。在此基础上,进一步研究了JHA对染毒与未染毒宿主消化生理的影响。结果表明,JHA处理不仅延长了6龄幼虫的寿命,增加了其取食量,而且还显著提高了幼虫的食物转化率和病毒产量。     

Protection of dogs from bites of phlebotomine sandflies by deltamethrin collars for control of canine leishmaniasis

Abstract. Dog collars made of PVC plastic impregnated with the pyrethroid insecticide deltamethrin at 40 mg/g were investigated for their protective efficacy against phlebotomine sandflies. Collared dogs were kept separately (two untreated control dogs lived together) in outdoor enclosures, each with a kennel, in the Cevennes, southern France. To measure sandfly mortality and anti-feeding effects due to the deltamethrin-impregnated collars worn continuously by the dogs for up to 8 months, each dog was periodically sedated and exposed for 2h to 150–200 laboratory-reared Phlebotomus perniciosus females (plus c. 25 males) inside a net (1.2m square, 1.8 m high) indoors. After dogs were removed from the nets, allowed to recover and returned to their kennels, any dead sandflies were collected from inside the net and counted. Surviving flies were kept overnight, then scored according to whether they were still alive or dead, unfed or blood-fed. From tests 2, 3, 4, 13, 20, 26 and 34 weeks after the dogs began wearing collars, the overall numbers of blood-fed female sandflies recaptured were 75 from two dogs with collars, compared with 1911 from two collarless dogs. Thus, for every 100 flies which fed on collarless dogs, only 4 fed on collared dogs, i.e. the collars protected dogs from 96% of the bites and this activity was maintained for up to 34 weeks. During the same period, the percentage of recaptured female sandflies that had fed on collared dogs was 0–12% compared to 55–95% on collarless dogs. Immediately after dogs were taken out of the nets, 21–60% of flies confined with the collared dogs were found dead, compared to 0–12% with the controls. It is concluded that, at least in the Mediterranean subregion, this insecticidal collar would protect a dog from the majority of sandfly bites and retain a killing effect for a complete sandfly season. Moreover, it seems likely that the use of collars on all dogs in a focus of Leishmania infantum would reduce contact between sandfly vectors and canine reservoir hosts sufficiently to diminish the risk of infection for humans as well as dogs.     

Studies on the behaviour and control of phlebotomine sandflies using experimental houses

Programmes for the control of phlebotomine sandflies (Diptera: Psychodidae), the vectors of leishmaniases, mainly target adults because larval breeding sites are generally unknown or inaccessible. To determine how blood‐questing sandfly females enter homes and to develop means for their control, an experimental house (EH) was constructed in a village endemic for cutaneous leishmaniasis. Initially, carbon dioxide (CO₂)‐baited suction traps were installed inside the EH to attract and capture sandflies. For other experiments, the windows of the EH were fitted with CO₂‐baited window entrance traps (WETs) that allow each window to be considered as a separate unit. The majority of captures inside the EH and in WETs consisted of Phlebotomus sergenti, a species that enters inhabited houses relatively infrequently. Analyses of collections in WETs and in sticky traps on external walls showed that sandflies entered windows having landed previously on the wall below or either side of the window. Shelves constructed below windows significantly reduced the numbers of sandflies that entered both the EH and inhabited houses. The lining of internal walls with insecticide‐impregnated fabric significantly increased mortality rates of sandflies captured inside the EH. To reduce the biting burden imposed by phlebotomine sandflies, several control measures must be integrated and sustained.     

Effects of juvenile hormone analogue treatment in adult females of the ovoviviparous cockroach,Blaptica dubia (Dictyoptera,Blaberidae)

Adult females of the ovoviviparous Argentinian cockroach, Blaptica dubia, were repeatedly treated with either 100?μg methoprene or 100?μg pyriproxyfen in 5?μL acetone either during the first vitellogenic cycle or during the period of gestation. Treatment during the first vitellogenic cycle (days 2–20 of adult life) did not inhibit vitellogenesis and oocyte growth, but prevented the formation of an ootheca. This was accompanied with a significant reduction of the titer of juvenile hormone (JH) III and an increased amount of ecdysone (E) and 20-hydroxyecdysone (20E) in the haemolymph of the animals. Treatment of adult females during the period of gestation (days 30–70) resulted in a complete degradation and resorption of the ootheca and induced another vitellogenic cycle. Again, this was associated with a decrease in haemolymph JH III titer, but an increase in the concentrations of free ecdysteroids.     

Regulation of juvenile hormone esterase activity in the European corn borer, Ostrinia nubilalis

The regulation of juvenile hormone esterase in last-instar diapause and nondiapause larvae of Ostrinia nubilalis was investigated using topically applied juvenile hormone I and a juvenile hormone mimic, methoprene. The influence of the head on juvenile hormone esterase was also investigated. Both juvenile hormone and methoprene caused increases in esterase levels when applied to feeding animals. Neither the hormone nor methoprene was capable of elevating nondiapause esterase activity to levels comparable to those found in untreated prediapause larvae. The esterase levels could be elevated in the larval body, without the head, during prepupal development of nondiapause larvae and in post-feeding diapause larvae. In both cases, juvenile hormone or methoprene induced juvenile hormone esterase activity in head-ligated animals. Topically applied methoprene prolonged feeding and delayed the onset of diapause. When methoprene was applied to larvae that had entered diapause, it disrupted diapause by inducing a moult.     

Juvenile hormone binding components of locust fat body

Juvenile hormone (JH) binding components from the fat body of the African migratory locust were analyzed in a search for a potential nuclear JH receptor. Biosynthetically prepared 10R[³H]JH III gave a high proportion of specific binding to isolated nuclei and extracted proteins; data obtained with the JH analogs, [³H]methoprene and [³H]pyriproxyfen, on the other hand, were obscured by abundant non-specific binding. The vast majority of the high affinity JH III binding activity present in cytosolic and nuclear extracts was due to a high molecular weight JH binding protein (JHBP) which has previously been identified in locust hemolymph. This protein has several chromatographic forms which interfered in the search for a nuclear JH receptor. When specific antiserum was used to remove JHBP from nuclear extracts, a novel JH binding activity (NBP) was detected. NBP could be separated from JHBP by precipitation with ammonium sulfate. NBP displayed a high affinity for JH III (Kd = 0.25 nM) and JH I and JH II competed strongly for JH III binding, whereas methoprene and pyriproxyfen showed apparent competition when present in 1,000-fold excess. NBP was present in nuclear extracts at approximately 25,000 sites per cell; levels were similar in male and female locusts and were not greatly affected by the presence or absence of JH. The characteristics of NPB make it a strong candidate for a nuclear JH receptor. © 1995 Wiley-Liss, Inc.     

Evaluation of deltamethrin-impregnated bednets and curtains against phlebotomine sandflies in Valle del Cauca, Colombia

Abstract. The effectiveness of bednets and curtains (nylon mesh 64 per cm²) impregnated with deltamethrin at 26 mg a.i./m² in reducing the biting nuisance caused by three phlebotomine sandfly species: Lutzomyia columbiana, Lu. lichyi and the predominant Lu. youngi (Diptera: Psychodidae), was evaluated at La Guaira, a rural settlement in Valle de Cauca near Cali, Colombia.
Pairs of volunteers collected sandflies under impregnated bednets, in rooms protected by impregnated curtains or in unprotected rooms in a randomized matched design. Collections were made in three houses per night on three consecutive nights, so that each house was sampled under each of the three treatments. This routine was repeated at 2-week intervals for 6 months. There was no significant difference between the overall numbers of sandflies collected in rooms with or without impregnated curtains. Only 0.14 sandflies/man-hour were caught on human bait under impregnated bednets, significantly fewer than the numbers collected on human bait outside the nets in the same room (1.91) or in unprotected rooms (3.29).
In a second set of experiments carried out in La Guaira and the neighbouring community of Jiguales, the effect of deltamethrin impregnation was evaluated by comparing numbers of sandflies collected on human bait under treated and untreated nets. Significantly fewer were collected under the impregnated nets (0.25 v. 0.69/man-hour). Wild-caught female Lu. youngi exposed to treated netting for 2 min in the laboratory all died with 24 h. The impact of deltamethrin-impregnated bednets was considered to be useful against Lu. youngi and other potential vectors of leishmaniasis in such communities.     

Mispatterning in the ommatidia of Apis mellifera pupae treated with a juvenile hormone analogue

To further understand the function of morphogenetic hormones in honeybee eye differentiation, the alterations in ommatidial patterning induced by pyriproxyfen, a juvenile hormone (JH) analogue, were studied by scanning and transmission electron microscopy. Prepupae of prospective honeybee workers were treated with pyriproxyfen and the effects on ommatidial differentiation were described at the end of the pupal development. The results show that the entire ommatidia, i.e., the dioptric as well as the receptor systems, were affected by the JH analogue. The wave of ommatidial differentiation, which progresses from the posterior to the anterior region of the pupal eyes, was arrested. In treated pupae, the rhabdomeres only differentiated at the apical axis of the retinula, the secondary and tertiary pigment cells did not develop their cytoplasm protrusions, and the cone cell quartet did not pattern correctly. Simultaneously, an intense vacuolization was observed in cells forming ommatidia. In a previous study we showed that pyriproxyfen exerts an inhibition on pupal ecdysteroid secretion. In this sense, the arrested ommatidial differentiation in pyriproxyfen-treated pupae could be due to a secondary effect resulting from an alteration in pupal ecdysteroid titers.     

Prospects for using pyroproxyfen-treated targets for tsetse control

Using¹⁴C cholesterol as a marker a positive correlation was established between the amount of oil (a chlorinated n-alkane containing 43–46% chlorine, ‘cereclor S45’) picked up by an adult tsetse fly exposed by tarsal contact to a treated surface and the duration of such exposure. Only a poor uptake was achieved from netting surfaces treated with less than 50% oil in acetone. Terylene netting treated with radioactive pyriproxyfen, [1-methyl-2-(4-phenoxyphenoxy)ethoxy] pyridine, dissolved in cereclor, was exposed in the field for a year. After 9 months 20% of the original radioactivity remained and was shown to be 95% authentic pyriproxyfen. Brief tarsal contact (up to 5 seconds) with such netting, by adult females ofGlossina morsitans morsitans Westwood, reduced the viability of their offspring to 28–43% of untreated control values. The effect was greatest in the reproductive cycle immediately following contact. Between 10 and 12 months after treatment of the fabric the radioactivity fell to only 7% of the original level but was associated mainly (>80%) with intact pyriproxyfen. Exposure of female flies to this netting resulted in a positive correlation between the duration of exposure and the extent of suppression of offspring viability, such that 2 min was sufficient to reduce offspring viability to zero for the life of the female. Traps or targets impregnated with conventional formulations of pyrethroids to kill tsetse would have lost all their activity by this time. Results are discussed in terms of the prospects for using pyriproxyfen-treated targets to sterilize female tsetse directly and also indirectly through the contamination of males prior to mating through contact with such targets.     

Effects of methoprene and juvenile hormone on larval ecdysis,emergence, and metamorphosis of the endoparasitic wasp, Apanteles congregatus

Topical application of juvenile hormone I and III or the hormone analogue methoprene to parasitized Manduca sexta larvae inhibited subsequent emergence of the endoparasitic wasp Apanteles congregatus. Methoprene treatment inhibited wasp emergence in a dose-dependent manner, causing either a delay or total inhibition of emergence. These results were interpreted as reflecting inhibitory effects of juvenile hormone on the second-larval ecdysis of the parasitoid that normally occurs during emergence from the host larva. Parasitoid ecdysis was disrupted even when methoprene was applied to host larvae a few hours prior to the normal expected time of emergence. A correlation between the number of emerging parasitoids and the timing of emergence was seen in methoprene-treated hosts, and few parasitoids emerged after day 9 of the host's fifth-instar. Our findings suggest that the suppression of emergence by juvenile hormone analogues noted in previous studies may be due to a similar inhibitory effect on parasitoid ecdysis. We also observed that parasitoids emerging from hosts treated with a low dose of methoprene (1 μg) later pupated normally but then formed nonviable pupal-adult intermediates. Thus use of this insect growth regulator must be undertaken carefully to prevent possible adverse effects on natural parasitoid populations.     

A novel geminal diol as a highly specific and stable in vivo inhibitor of insect juvenile hormone esterase

Thio-containing and acetylenic trifluoromethyl ketones were potent inhibitors of insect juvenile hormone (JH) esterase with greater inhibitory activity than aliphatic and α,β-unsaturated homologs. Octylthio-1,1,1-trifluoropropan-2-one was the most potent inhibitor with the greatest equilibrium hydration constant in pure water. However, a keto/hydrate equilibrium was not necessary for JH esterase inhibition. The carbonyl tautomer of 1-octyl [1-(3,3,3-trifluoropropan-2,2- dihydroxy)] sulfone (OTPdOH-sulfone) was not detectable, and yet OTPdOH-sulfone was a potent in vitro inhibitor of JH esterase with an I₅₀ of 1.2 nM. The mechanism of JH esterase inhibition by these compounds is discussed. OTPdOH-sulfone inhibited JH esterase with minimal activity toward insect 1-naphthyl acetate esterase and electric eel acetylcholinesterase. The inhibitor was also active in vivo, selective for JH esterase, and persistent for over 32 h. OTPdOH-sulfone when topically applied to larval and adult cabbage loopers, Trichoplusia ni, elicited juvenoid activity apparently because of the specific in vivo inhibition of JH metabolism. Arch. Insect Biochem. Physiol. 36:165–179, 1997. © 1997 Wiley-Liss, Inc.     

Evaluation of light-emitting diodes as attractant for sandflies (Diptera: Psychodidae: Phlebotominae) in northeastern Brazil

Hoover Pugedo light traps were modified for use with green and blue-light-emitting diodes to trap phlebotomine sandflies in northeastern Brazil. A total of 2,267 specimens belonging to eight genera and 15 species were sampled. The predominant species were Nyssomyia whitmani(34.41%) and Micropygomyia echinatopharynx(17.25%).The green LED trap prevailed over the blue and control lights; however, no statistically significant difference could be detected among the three light sources. Even without statistical significance, we suggest using LEDs as an attractant for the capture of sandflies because of several advantages over the conventional method with incandescent lamps.     

Growth of the male accessory gland in adult locusts: Roles of juvenile hormone,JH esterase,and JH binding proteins

The participation of juvenile hormone (JH) in the regulation of growth and protein synthesis in the accessory reproductive gland of male Locusta migratoria has been investigated. After elimination of endogenous JH with ethoxyprecocene, the accessory gland failed to grow, but growth was restored by a single application of the JH analog, pyriproxyfen. Pyriproxyfen appeared to stimulate total protein synthesis by 3 h, with a significant effect by 12 h, in contrast to 24 h observed in fat body. The dose curve for stimulation of protein synthesis 12 h after applying pyriproxyfen gave an ED₅₀ of 0.1 μg; the dose curve for gland growth at 72 h was biphasic, with steps at about 0.01 μg and 10 μg, suggesting two phases in JH action. SDS-PAGE analysis showed several components that were stimulated by pyriproxyfen, the effect being strongest in an 11 kDa band. A 5 kDa component was enhanced in the soluble and reduced in the particulate fraction after precocene treatment. The accessory gland contained JH esterase activity at levels about 100 times those in fat body or hemolymph, and was higher in precocene treated locusts. Binding activity for [³H]10R -JH III was high in cytosolic and nuclear fractions, and was identified immunologically as due to the previously described hemolymph JH binding protein. The results indicate that the mode of action of JH in the accessory gland may differ from that in the fat body. The presence of intracellular JH binding protein suggests a direct action of JH within the gland, that may be modulated by JH esterase. © 1995 Wiley-Liss, Inc.     

Effects of experience and juvenile hormone on the organization of the mushroom bodies of honey bees

There is an age-related division of labor in the honey bee colony that is regulated by juvenile hormone. After completing metamorphosis, young workers have low titers of juvenile hormone and spend the first several weeks of their adult lives performing tasks within the hive. Older workers, approximately 3 weeks of age, have high titers of juvenile hormone and forage outside the hive for nectar and pollen. We have previously reported that changes in the volume of the mushroom bodies of the honey bee brain are temporally associated with the performance of foraging. The neuropil of the mushroom bodies is increased in volume, whereas the volume occupied by the somata of the Kenyon cells is significantly decreased in foragers relative to younger workers. To study the effect of flight experience and juvenile hormone on these changes within the mushroom bodies, young worker bees were treated with the juvenile hormone analog methoprene but a subset was prevented from foraging (big back bees). Stereological volume estimates revealed that, regardless of foraging experience, bees treated with methoprene had a significantly larger volume of neuropil in the mushroom bodies and a significantly smaller Kenyon cell somal region volume than did 1-day-old bees. The bees treated with methoprene did not differ on these volume estimates from untreated foragers (presumed to have high endogenous levels of juvenile hormone) of the same age sampled from the same colony. Bees prevented from flying and foraging nonetheless received visual stimulation as they gathered at the hive entrance. These results, coupled with a subregional analysis of the neuropil, suggest a potentially important role of visual stimulation, possibly interacting with juvenile hormone, as an organizer of the mushroom bodies. In an independent study, the brains of worker bees in which the transition to foraging was delayed (overaged nurse bees) were also studied. The mushroom bodies of overaged nurse bees had a Kenyon cell somal region volume typical of normal aged nurse bees. However, they displayed a significantly expanded neuropil relative to normal aged nurse bees. Analysis of the big back bees demonstrates that certain aspects of adult brain plasticity associated with foraging can be displayed by worker bees treated with methoprene independent of foraging experience. Analysis of the over-aged nurse bees suggests that the post-metamorphic expansion of the neuropil of the mushroom bodies of worker honey bees is not a result of foraging experience. © 1995 John Wiley & Sons, Inc.     

Critical roles for juvenile hormone and its esterase+ in the prepupa of Trichoplusia ni

In the caterpillar Trichoplusia ni (Lepidoptera: Noctuidae) it has been demonstrated by allatectomy that the appearance of juvenile hormone during the prepupal stage is crucial for the successful larval-pupal ecdysis of most larvae. Application of juvenile hormone or juvenile hormone esterase inhibitors at key times disrupted normal development as well. Thus the subsequent disappearance of juvenile hormone is regulated by degradation by juvenile hormone esterase in addition to a hypothetical reduction in biosynthesis. This reduction in juvenile hormone titer in the prepupa is just as critical for normal development as was its previous appearance. These observations on the critical role of juvenile hormone in the prepupa are in contrast to observations in some other species. For instance, in the case of Manduca sexta (Lepidoptera: Sphingidae), juvenile hormone is considered only supplementary to the action of prothoracicotropic hormone in the postwandering stage and primarily is required for normal pupal development. It thus appears that even within the Lepidoptera the role of juvenile hormone in prepupal development can vary dramatically.     

The effects of juvenile hormone, 20-hydroxyecdysone and precocene II on activity of corpora allata and the mode of negative-feedback regulation of these glands in the adult Colorado potato beetle

When the titre of juvenile hormone III in female Leptinotarsa decemlineata was elevated by the implantation of supernumerary corpora allata or by the injection of the hormone, the rate of endogenous hormone production by the host glands was significantly restrained, as determined by the short-term in vitro radiochemical assay. From denervation studies, it is suggested that during phases of elevated juvenile hormone titre, the corpus allatum activity is regulated via humoral as well as neural factors requiring intact nerve connections. Restrainment of gland activity appears to be mainly via the neural pathway. Isolated corpora allata were not influenced by 10^?5 M juvenile hormone III added to the incubation medium in vitro.Studies with farnesenic acid revealed that the final two enzymatic steps in the biosynthetic pathway of juvenile hormone are also diminished during prolonged neural inhibition of the corpora allata.20-Hydroxyecdysone and precocene II had no apparent effect on the corpus allatum activity of Leptinotarsa decemlineata.