Significance of penetration and metabolism on topical toxicity of diflubenzuron in Spodoptera littoralis and Spodoptera exigua

Toxicity assays indicated that when diflubenzuron (DFB) was topically applied on larvae of the Egyptian cotton leafworm, Spodoptera littoralis (Boisduval), and the beet armyworm, Spodoptera exigua (Hübner), estimated LD₅₀ values were similar, reaching 0.47 and 0.44 ng/larva, respectively. This study evaluated the importance of the rate of uptake and excretion, and of enzymatic metabolism in building up an insecticidal toxicity after topical application on the insect cuticle in both species. In general, penetration of DFB in S. littoralis was about 2-fold higher than in S. exigua, whereas metabolic breakdown was of minor importance in S. littoralis as compared with S. exigua. The current data indicate the importance of cuticle penetration and discuss that extrapolation from oral feeding toxicity results to topical toxicity data should be evaluated with caution, especially for insect growth regulators used in integrated pest management.     

USE OF BLUE LAKE AS AN INDICATOR OF BACTERIAL PENETRATION INTO EGGS

Blue Lake, an insoluble dye, was evaluated as an indicator of potential bacterial penetration into eggs. Various groups of eggs (fresh-laid; commercial; water-washed) were dipped in 0.25% (w/v) Blue Lake in 0.1% Triton X-100 solution for 2 min and incubated at room temperature up to 1 ± 24 h. Penetration was detected by counting the blue dots on shell membranes after breaking the eggs. Commercial eggs allowed the easiest penetration. All commercial eggs showed blue dots even at 2 min incubation and the average count reached 111/egg at 1 h. Water-washed eggs allowed much less penetration than commercial eggs and the counts of blue dots on those eggs were 10/egg at 2 min and 22 at 1 h. Fresh-laid eggs did not allow any penetration up to 24 h. Above results corresponded very well with the penetration study with Salmonella enteritidis and also with the morphological study of eggshell surfaces using electron microscopy where fresh-laid eggs had intact cuticle layers, but commercial eggs did not. Thus, Blue Lake dye might be used as a rapid indicator of bacterial penetration through eggshells.     

Genomic and proteomic studies on the effects of the insect growth regulator diflubenzuron in the model beetle species Tribolium castaneum

Several benzoylphenyl urea-derived insecticides such as diflubenzuron (DFB, Dimilin) are in wide use to control various insect pests. Although this class of compounds is known to disrupt molting and to affect chitin content, their precise mode of action is still not understood. To gain a broader insight into the mechanism underlying the insecticidal effects of benzoylphenyl urea compounds, we conducted a comprehensive study with the model beetle species and stored product pest Tribolium castaneum (red flour beetle) utilizing genomic and proteomic approaches. DFB was added to a wheat flour-based diet at various concentrations and fed to larvae and adults. We observed abortive molting, hatching defects and reduced chitin amounts in the larval cuticle, the peritrophic matrix and eggs. Electron microscopic examination of the larval cuticle revealed major structural changes and a loss of lamellate structure of the procuticle. We used a genomic tiling array for determining relative expression levels of about 11,000 genes predicted by the GLEAN algorithm. About 6% of all predicted genes were more than 2-fold up- or down-regulated in response to DFB treatment. Genes encoding enzymes involved in chitin metabolism were unexpectedly unaffected, but many genes encoding cuticle proteins were affected. In addition, several genes presumably involved in detoxification pathways were up-regulated. Comparative 2D gel electrophoresis of proteins extracted from the midgut revealed 388 protein spots, of which 7% were significantly affected in their levels by DFB treatment as determined by laser densitometry. Mass spectrometric identification revealed that UDP-N-acetylglucosamine pyrophosphorylase and glutathione synthetase were up-regulated. In summary, the red flour beetle turned out to be a good model organism for investigating the global effects of bioactive materials such as insect growth regulators and other insecticides. The results of this study recapitulate all of the different DFB-induced symptoms in a single model insect, which have been previously found in several different insect species, and further illustrate that DFB treatment causes a wide range of effects at the molecular level.     

Eggshell pigmentation has no evident effects on offspring viability in common kestrels

Infectious diseases and parasitism are major environmental forces decreasing fitness, and thus individual strategies aimed at preventing pathogen infections, either in an individual or their offspring, should be favoured by natural selection. The mineral fraction and some organic compounds in the shells of bird eggs are considered physical and chemical barriers against pathogen penetration to the embryo. However, eggshell pigment deposition has only recently been considered as a mechanism to resist pathogen penetration into the egg. By wiping the eggshell surface, the amount of pigment and some cuticle proteins were experimentally manipulated for the first time in nature. The effects on egg hatchability and offspring viability measured as nestling condition, immunocompetence and probability of recruitment were investigated in the common kestrel Falco tinnunculus. Protoporphyrin IX and biliverdin IXα to a lesser extent were the only identified pigments. The concentration of protoporphyrin IX and cuticle proteins were significantly reduced in the wiped with respect to the control treatment. Our study shows no evidence of a detrimental effect of the reduction of eggshell pigments on egg hatchability, mortality of the chicks during the nesting period, nestling body condition, nestling local immune response to PHA antigen or probability of recruitment. Further research will be necessary to elucidate the direct role of protoporphyrins and other pigments in egg bacterial infection.     

Action and pharmacokinetics of a novel insect growth regulator,halofenozide, in adult beetles of Aubeonymus mariaefranciscae and Leptinotarsa decemlineata

Topical application of halofenozide on adults of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and Aubeonymus mariaefranciscae (AMF) exhibited its effects in a different way in the two coleopterans. In CPB, the fecundity was strongly affected, whereas the main effect in AMF was a drastic decrease of the progeny survival. The rate of penetration of labeled halofenozide through the cuticle followed a similar pattern in CPB and AMF, whereas the rate of excretion was much more rapid in AMF. The extremely slow excretion of the product in CPB suggested that this fact must contribute to the prolonged action of halofenozide on fecundity at its highest dose of 20 μg per adult. Retention of halofenozide in the reproductive system of males of AMF and CPB was low, whereas high levels of radioactivity were recovered in females (ovaries + eggs). Comparing females of both coleopterans, CBP retained a higher amount than AMF. Moreover, treated CPB adults showed a rapid cessation of oviposition due to distorted ovaries, detrimental oocyte growth, and loss of oviposition due to the fact that yolk protein synthesis and/or incorporation into eggs was reduced. In AMF, the development of the progeny was strongly affected, and more than 80% of first‐instar larvae died after egg hatching in the first 25 days after the treatment, due to premature molting and inhibition of ecdysis. Cross sections of intoxicated first larval instars of AMF originating from adults that were topically treated with halofenozide showed the presence of a double cuticle and these larvae could not shed the old cuticle. Arch. Insect Biochem. Physiol. 41:201–213, 1999. © 1999 Wiley‐Liss, Inc.     

Cuticular encystment of Autographa nigrisigna eggs by epidermal cell migration

It was previously established that Autographa nigrisigna loopers form cuticular cysts at the dorsal site of the 9th (penultimate) abdominal segment after parasitization by the solitary endoparasitoid Campoletis chlorideae and get rid of the parasitoid egg with the old cuticle at ecdysis. The cuticular cyst consists of a space between the old cuticle and new cuticle formed by the epidermis to enclose the parasitoid egg. The fact that A. nigrisigna loopers exclude the oviposited egg from the hemocoel using a cuticular cyst raises the question how the parasitoid egg passes through the epidermis. To exclude the endoparasitoid eggs from the hemocoel, the epidermis is required to move the location of the parasitoid egg. In the current study, we investigated the morphological process of cuticular cyst formation. First, the oviposited egg drifted to the 9th abdominal segment located at the open end of the dorsal vessel as a result of force generated by the hemolymph current from the oviposition site, and formed contacts with the integument containing the fat body (FB). The epidermis, in contact with the egg, then began to move along with the basement membrane formed on the surface of the FB, and settled under the egg, thus altering its location. This inversion was duplicated in vitro using integument from the 9th abdominal segment when parasitoid eggs were inserted between the epidermis and FB. When the integument, without the FB, was incubated on an agar plate, the epidermal cells migrated on the plate. Integument without eggs showed no signs of migration from their original sites. When the actin polymerization inhibitor latrunculin B was added to the cultures, the epidermal cells remained in their original location.     

Egg marking in the facultatively queenless ant Gnamptogenys striatula: the source and mechanism

Conflicts over reproductive division of labour are common in social insects. These conflicts are often resolved via antagonistic actions that are mediated by chemical cues. Dominant egg layers and their eggs can be recognized by a specific yet similar cuticular hydrocarbon profile. In the facultatively queenless ant Gnamptogenys striatula, a worker's cuticular hydrocarbon profile signals its fertility and this determines its position in the reproductive division of labour. How eggs acquire the same hydrocarbon profile is as yet unclear. Here, we search for glandular sources of egg hydrocarbons and identify the putative mechanism of egg marking. We found that eggs carry the same hydrocarbons as the cuticle of fertile workers, and that these hydrocarbons also occur in the ovaries and the haemolymph. None of the studied glands (Dufour, venom, labial and mandibular gland) contained these hydrocarbons. Our results indicate that hydrocarbons are deposited on eggs while still in the ovaries. The low hydrocarbon concentration in the ovaries, however, suggests they are produced elsewhere and transported through the haemolymph. We also found that fertile workers regularly deposit new hydrocarbons on eggs by rubbing laid eggs with a hairy structure on the abdominal tip from which a non-polar substance is secreted.     

Compatibility of Spinosad, Tebufenozide and Azadirachtin with Eggs and Pupae of the Predator Chrysoperla carnea (Stephens) Under Laboratory Conditions

Under laboratory conditions, the toxicity of three novel insecticides, spinosad (Tracer ® ), tebufenozide (Mimic ® ) and azadirachtin (Align ® ), was tested against eggs and pupae of the predator Chrysoperla carnea (Stephens). In a first series of assays, eggs were dipped in an aqueous concentration and no ovicidal activity was scored for the three insecticides. In the second, when females were ovipositing on treated substrate for 24 h, fecundity and hatching percentages were similar as compared to controls and the offspring developed normally until the adult stage. However, spinosad, at the highest concentrations tested, caused a slight, significant reduction in the adult life span and fecundity. In a third series of experiments, pupae developed into normal adults after topical treatment for the three insecticides. Herewith, a pharmacokinetic study indicated low accumulation in the body after pupal cuticle penetration when administrating 14 C-labelled insecticide. Fourthly, pupation of last-instar larvae in treated substrate was normal for spinosad and tebufenozide. Only azadirachtin caused a slight reduction in the number of pupae and adults; however, fecundity and fertility of surviving adults was normal. In conclusion, the current results indicate that the three insecticides are not toxic to eggs and pupae of C. carnea .     

Artificial miniaturization causes eggs laid by crowd-reared (gregarious) desert locusts to produce green (solitarious) offspring in the desert locust, Schistocerca gregaria

The mechanism underlying the phase-dependent polyphenism in hatchling body coloration was studied by testing for a possible causal relationship with egg size in the desert locust, Schistocerca gregaria. Crowd-reared (gregarious) females typically produce large, black offspring, whereas females reared in isolation (solitarious) deposit small, green offspring. We first tested for possible genetic differences in the role of egg foam by washing or separating eggs from two strains of locust. No solitarizing effect was found in either of the strains tested, supporting a previous finding, using another laboratory strain, to show that the hatchling body coloration and size are pre-determined in the ovary of the mother and no egg foam factor is involved in the control of the hatchling body coloration. Topical application of fenoxycarb, a juvenile hormone analog (JHA), and implantation of extra corpora allata (CA), taken from Locusta migratoria, caused gregarious female adults of S. gregaria to produce small eggs. Some eggs laid by CA-implanted females produced green hatchlings. All large eggs chosen among those deposited by gregarious females produced black hatchlings. When eggs were either kept on dry filter paper at nearly saturated relative humidity during embryogenesis or pricked with a needle so that some egg yolk was squeezed out, some produced small, green hatchlings. These results suggested that the amount of egg yolk or the availability of yolk material may determine the body coloration of hatchlings.     

Penetration of zona-free hamster eggs by liposome-treated sperm from the bull, ram, stallion, and boar

Spermatozoa from each of four rams, four stallions, and three boars (six semen samples) were treated with dilauroylphosphatidylcholine (PC12) liposomes and compared with control bull sperm to induce the acrosome reaction (AR) and study possible penetration of the sperm into zona-free hamster eggs. Diluted sperm were incubated with several concentrations of PC12 for 7 min at 39 degrees C prior to insemination of the hamster eggs in vitro. The sperm from the bull were diluted to 10(6) cells/ml, as previously studied. Sperm from the ram, stallion, and boar were diluted to 6 X 10(6) and 20 X 10(6) cells/ml. After addition to the eggs, the sperm concentration was reduced by 75 percent. Inseminated eggs were incubated with sperm for 3 h at 39 degrees C prior to being fixed, stained, and observed for sperm penetration. At an initial concentration of 6 X 10(6) cells/ml, bull sperm treated with 36.7 microM PC12 achieved an egg penetration rate of 92%, whereas under nearly identical conditions stallion spermatozoa achieved only 54% egg penetration. Under similar conditions, ram spermatozoa failed to penetrate eggs, but when the initial sperm concentration was increased to 20 X 10(6) cells/ml, sperm incubated with 51.1 microM PC12 achieved 52% egg penetration. Boar spermatozoa treated with PC12 at either sperm concentration failed to exhibit an AR or penetrate hamster eggs. In general, as PC12 concentration increased the percentage of sperm with an AR increased and sperm motility decreased. It is concluded that 1) PC12 liposomes are effective in inducing the AR in sperm from the bull, ram, and stallion, but under conditions tested are ineffective with boar sperm;(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of rates of penetration through insect cuticle of amphiphylic analogs of insect pyrokinin neuropeptides

Rates of penetration through the cuticle of amphiphylic analogs, synthesized by addition of 6-phenylhexanoic acid or 9-fluoreneacetic acid or 1-pyrenebutyric acid to the amino terminus of the pentapeptide Phe-Thr-Pro-Arg-Leu-amide, were assessed by quantitative analysis using reversed phase liquid chromatography. The analogs effectively penetrated the cuticle of both the adult American cockroach and tobacco budworm moth. However, the amounts of analogs that penetrated the cuticle of the cockroach were significantly lower and the rates of penetration were slower than for moth cuticle. Penetration of the analogs through the cuticle was dependent upon the size of the lipidic attachment to the pentapeptide. The 6-phenylhexanoic acid analog penetrated most rapidly followed by the 9-fluoreneacetic acid analog and the 1-pyrenebutyric acid analog penetrated slowest. All of the analogs exhibited an initial rapid period of penetration lasting 2-3 h followed by the establishment of a steady slow release state which lasted between 9-24 h and was dependent upon both the size and surface area of the aromatic lipidic portion of the analog and species of insect to which the analog was applied. The results confirmed the hypothesis that the insect cuticle could be employed as a slow release device for delivery of analogs of insect neuropeptides.     

寄主钾营养对烟粉虱发育、存活和寄主选择的影响

为了探讨寄主钾营养对烟粉虱发育、存活和寄主选择的影响,设置了K0（0 mg/L）、K30（30 mg/L）、K60（60 mg/L）、K120（120 mg/L）和K240（240 mg/L）5种不同钾浓度,研究了烟粉虱在不同处理黄瓜上的发育历期、存活率、体形以及成虫的寄主选择。结果表明：在温度（26±1）℃,相对湿度80%±5%的条件下,取食不同钾营养水平黄瓜的烟粉虱在发育历期、存活率、体形上均有差异,其中在K240处理黄瓜上烟粉虱若虫发育最慢,从卵到成虫的时间最长,为21.4天,而在K60处理黄瓜上最快,为18.3天。在不同钾浓度处理寄主上从卵到成虫存活率以K30处理黄瓜上最高（84.7%）,其次依次为K60（83.8%）、K120（76.2%）、K0（71.4%）和K240（64.8%）。在体形上以K30处理黄瓜上最大,K240处理黄瓜上最小。寄主选择性试验结果表明,在温室条件下,烟粉虱成虫喜欢在K60处理黄瓜上取食和产卵。这些结果提示在一定范围内可以通过调节寄主钾营养状况来调控烟粉虱种群。     

Mothers influence offspring body size through post-oviposition maternal effects in the redbacked salamander, Plethodon cinereus

In the terrestrial salamander (Plethodon cinereus), previous work has shown that mothers body size is positively correlated to offspring size at the time of hatching even after controlling for the effects of egg size. This study was designed to determine whether maternal body size affects offspring size via pre-oviposition factors (e.g., yolk quality, jelly coat composition, or maternal genes) or post-oviposition factors (e.g., parental care behaviors, parental modification of environment). Gravid females were captured and induced to lay eggs in experimental chambers in which the environment was standardized. Fifteen clutches were exchanged, or cross-fostered, between female pairs differing in body size. Ten females whose eggs were taken away and then returned served as controls for the crossing treatment. Foster mothers did not significantly differ from control mothers in the time spent with eggs, body position, or number of egg movements during brooding. Average egg mass measured midway through development was not significantly correlated to the body size of either the genetic or foster mother, but was correlated to pre-oviposition oocyte size. At hatching, offspring body length was positively correlated to egg size and the foster mothers body size. This correlation suggests that in P. cinereus post-oviposition maternal effects have a greater impact on offspring size than other maternal factors incorporated into the egg prior to oviposition. While our study showed that larger mothers moved their eggs less often and tended to spend more time in contact with their eggs, further work needs to be done to identify the specific mechanisms through which larger mothers influence the body size of their offspring. This is the first experimental demonstration of post-oviposition maternal effects for any amphibian with parental care.     

Diflubenzuron-Induced alterations during in vitro development of Tenebrio molitor pupal integument

The effects of diflubenzuron (DFB) in Tenebrio molitor pupae were first investigated on cuticle secretion induced by 20-hydroxyecdysone in vitro. The sternal integuments were treated by DFB either 3 days before culture or during culture. DFB, when applied before culture, did not prevent the molting hormone from inducing a new cuticle deposition by integument explants in vitro. However, this cuticle showed several architectural alterations and a thickness reduction. When applied during the culture in the presence of 20-hydroxyecdysone, DFB at high dose (≥ 20 μg/ml) was able to inhibit cuticle secretion, but lower doses (? 10 μg/ml) resulted in epicuticle deposition. These observations confirm in vivo studies showing antagonistic effects of DFB and ecdysteroids at the level of epidermal cells. In another series of experiments, the DFB effects were analyzed without addition of exogenous molting hormone in vitro. Because it had been observed in previous studies that pupal epidermal explants of Tenebrio secrete low but significant amounts of ecdysteroids in the culture medium, this in vitro secretion was measured by radioimmunoassay after DFB treatment. It was observed that DFB, when applied either before or during culture, significantly reduced the hormonal secretion in vitro. This reduction, observed at the level of epidermal cells, could be homologous with the diminution of the endogenous ecdysteroid peak previously described after in vivo DFB treatment in Tenebrio pupae.     

Carbohydrate metabolism and restricted oxygen supply in the eggs of the silkworm, Bombyx mori

Increased oxygen supply to diapause eggs of the silkworm (O₂-incubation) effectively prevented diapause initiation and induced the same pattern of glycogen, polyol and lactate levels as was observed in normal non-diapause eggs. Sensitivity to oxygen decreased as embryonic development proceeded. After the termination of this sensitive period, accumulation of polyols and lactate followed.Experiments were carried out to test whether changes in the oxygen permeability of the egg membranes are involved in restricting the supply of this gas to eggs at the onset of diapause. Oxygen permeability of the chorion was measured with apparatus especially designed for this purpose. Although the chorion of the diapause egg was less permeable than that of the non-diapause egg, the oxygen permeability of the chorion does not change appreciably during the early developmental stages of the diapause eggs. The changes in rate of water loss through the egg membranes were measured during the early developmental stages of the embryos. The level of water loss decreased gradually as the formation of serosal cuticle proceeded. Moreover, it was observed that the water loss up to the time of formation of serosal cuticle was closely related to the oxygen permeability of the chorion.From these results, we suggest that the formation of the serosal cuticle may be an additional cause of the restricted oxygen supply at the onset of the diapause.     

Damage,repair and regeneration in insect cuticle: The story so far,and possibilities for the future

The exoskeleton of an insect can contain countless specializations across an individual, across developmental stages, and across the class Insecta. Hence, the exoskeleton's building material cuticle must perform a vast variety of functions. Cuticle displays a wide range of material properties which are determined by several known factors: the amount and orientation of the chitin fibres, the constituents and degree of cross-linking and hydration of the protein matrix, the relative amounts of exo- and endocuticle, and the shape of the structures themselves. In comparison to other natural materials such as wood and mammal bone, relatively few investigations into the mechanical properties of insect cuticle have been carried out. Of these, very few have focussed on the need for repair and its effectiveness at restoring mechanical stability to the cuticle. Insect body parts are often subject to prolonged repeated cyclic loads when running and flying, as well as more extreme “emergency” behaviours necessary for survival such as jumping, wedging (squeezing through small holes) and righting (when overturned). What effects have these actions on the cuticle itself? How close to the limits of failure does an insect push its body parts? Can an insect recover from minor or major damage to its exoskeleton “bones”? No current research has answered these questions conclusively.     

Pathogenicity of entomopathogenic hyphomycetes, Paecilomyces fumoso-roseus and Nomuraea rileyi, to eggs of noctuids, Mamestra brassicae and Spodoptera littoralis

Bioassays were conducted to determine the susceptibility of egg masses of Mamestra brassicae and Spodoptera littoralis to different spore doses of Paecilomyces fumoso-roseus and Nomuraea rileyi at 20° and 25°C. P. fumoso-roseus was highly virulent against eggs, whereas N. rileyi provoked only a deferred mortality of larvae hatched from treated eggs. Nevertheless, larval mortality of S. littoralis caused by N. rileyi at 25°C was more effective after first-instar larval contamination than after egg mass treatment. The duration of the egg stage could explain differences of susceptibility between the two noctuids at 25°C. Scanning electron microscopical observations suggested two ways of contamination of newly hatched larvae. First, fungal germinations on the chorion surface suggested that newly hatched larvae might be infected by penetration of the egg integument before hatching. Second, conidia on the egg cuticle could be an entomopathogenic inoculum for newly emerging larvae which fed upon chorions. Results showed that pathogenicity of Hyphomycetes to noctuid eggs might be a promising area of investigation for biological control.     

Annual follow-up of the gastrointestinal parasitosis of the tepezcuintle,Agouti paca (Rodentia: Agoutidae) in captivity in the Mexican tropic

Previous reports showed that the tepezcuintle (Agouti paca) is commonly infested by gastrointestinal parasites (GIP), mainly Eucoccidiida and helminths. However, there is no available information on the frequency of those parasites and their faecal egg excretions at different moments during the year. These information would provide a valuable baseline for the establishment of control strategies against GIP in tepezcuintles under captivity. The objectives of the present study were to determine the prevalence of GIP orders and genera that infest tepezcuintles under captivity and, to describe the dynamics of faecal egg and oocyst excretion in a year. Ten tepezcuintles were sampled (faeces) twice every month for twelve months. The faecal samples were processed by the flotation and McMaster techniques. Two orders of parasites were determined: Strongylida and Eucoccidiida. Two genera of nematodes were also determined: Strongyloides and Trichuris. The prevalence of Strongylida eggs, Eucoccidiida oocysts and Trichuris sp. eggs reached 10-20% of animals in certain months. The most important genus was Strongyloides, found in 60 to 100% of the animals year round. The average excretion of eggs in the group was 45 to 372 eggs per gram. Tepezcuintles kept under captivity in Yucatan are parasited with Strongyloides sp throughout the year, but only occasionally had oocysts of Eucoccidiida and eggs of Strongylida and Trichuris sp.