Physiological and morphological aspects of Aedes aegypti developing larvae: effects of the chitin synthesis inhibitor novaluron

Population control of the dengue vector mosquito, Aedes aegypti, is difficult due to many reasons, one being the development of resistance to neurotoxic insecticides employed. The biosynthesis of chitin, a major constituent of insect cuticle, is a novel target for population control. Novaluron is a benzoylphenylurea (BPU) that acts as a chitin synthesis inhibitor, already used against mosquitoes. However, information regarding BPU effects on immature mosquito stages and physiological parameters related with mosquito larval development are scarce. A set of physiological parameters were recorded in control developing larvae and novaluron was administered continuously to Ae. aegypti larvae, since early third instar. Larval instar period duration was recorded from third instar until pupation. Chitin content was measured during third and fourth instars. Fourth instars were processed histochemically at the mesothorax region, stained with hematoxylin and eosin (HE) for assessment of internal tissues, and labeled with WGA-FITC to reveal chitinized structures. In control larvae: i) there is a chitin content increase during both third and fourth instars where late third instars contain more chitin than early fourth instars; ii) thoracic organs and a continuous cuticle, closely associated with the underlying epidermis were observed; iii) chitin was continuously present throughout integument cuticle. Novaluron treatment inhibited adult emergence, induced immature mortality, altered adult sex ratio and caused delay in larval development. Moreover, novaluron: i) significantly affected chitin content during larval development; ii) induced a discontinuous and altered cuticle in some regions while epidermis was often thinner or missing; iii) rendered chitin cuticle presence discontinuous and less evident. In both control and novaluron larvae, chitin was present in the peritrophic matrix. This study showed quantitatively and qualitatively evidences of novaluron effects on Ae. aegypti larval development. To our knowledge, this is the first report describing histological alterations produced by a BPU in immature vector mosquitoes.     

The effects of Plagiorchis noblei metacercariae on the development and survival of Aedes aegypti larvae in the laboratory

Plagiorchis noblei infections impair the survival and development of fourth instar Aedes aegypti larvae. Mortality during the larval and pupal stages reached 92%, and 60% of the emerging adults were malformed. The metacercariae interfere with pupation and the emergence of adults. Larvae and pupae that fail to transform to the next developmental stage within the normal time characteristically persist for extended periods, but invariably die without transforming. Whereas 82% of the control larvae gave rise to functional adults, only 4% of infected larvae managed to do so. Such effects may facilitate the transmission of the parasite.     

敲减神经肽F基因(npf)对亚洲玉米螟取食、生长发育和繁殖的影响

【目的】神经肽F(neuropeptide F, NPF)是无脊椎动物特有的一类神经肽,因其C末端是苯丙氨酸(F)而命名,参与昆虫的取食、生物节律、学习记忆等多种生理功能的调控。本研究旨在明确NPF对亚洲玉米螟Ostrinia furnacalis生长发育的影响,为害虫防治提供重要依据。【方法】采用一种基于工程菌高效合成靶向昆虫基因的dsRNA的方法经济有效地敲降npf,用低浓度(0.01%)和高浓度(0.02%)dsNPF和dsGFP(对照)分别饲喂亚洲玉米螟1龄初、3龄初和5龄初幼虫直至化蛹,检测5龄幼虫平均取食量、体重、体长、存活率和化蛹率,蛹羽化率和成虫产卵量,以及幼虫各龄期、蛹发育历期和成虫寿命。【结果】从亚洲玉米螟1, 3和5龄初幼虫开始饲喂0.01%和0.02% dsNPF时,与饲喂相应浓度dsGFP的对照相比,除个别点外,5龄幼虫的取食量、体重、体长、存活率和化蛹率,蛹羽化率和成虫单雌产卵量均显著降低,幼虫各龄期、蛹发育历期均显著延长,成虫寿命显著缩短。且dsNPF处理幼虫的龄期越早对发育的影响越大。其中0.01% dsNPF处理的1龄幼虫和0.02% dsNPF处理的3龄幼虫有90%的个体在蛹期死亡,而0.02%dsNPF处理的1龄幼虫有90%的个体在幼虫期死亡。【结论】结果提示NPF对亚洲玉米螟的发育和取食具有调控作用,这为探索新型绿色的害虫防治提供了依据。     

Life history traits of Aedes caspius (Diptera: Culicidae): a laboratory study of larval stages

The larval survival and development times of Aedes caspius (Diptera: Culicidae) were examined in the laboratory. These life history traits were estimated using life tables constructed for two populations, one of which had been subjected to a long-term larvicide control program. Traits were evaluated for eight different population densities. The effects of population, larval stage and larval density were investigated using a general linear model. Density was positively correlated with larval survival but did not affect development time. The fourth instar and pupae had the lowest larval survival rates. First and fourth instar larvae had the longest development times. These traits were not significantly different between the two populations. The effect of larvicide control on these traits is discussed.     

The effects of pH,phenol, and sodium chloride on survival and caloric,lipid, and nitrogen content of a laboratory population of chironomus attenuatus (Walk.)

Continuous flow, laboratory microcosms were used to measure the effects of pH, phenol, and NaCl on the survival of the life stages of Chironomus attenuatus, the caloric content of third and fourth instar larvae and adults, the lipid and protein-nitrogen content of fourth instar larvae, and the interaction among the various responses. The metabolism of phenol was studied using uniformly labeled phenol-C¹⁴. pH had a significant effect on the survival of all life stages of C. attenuatus. Survival was higher for all larval instars at pH 7.2 while adult emergence was higher at pH 6.2. Increasing phenol levels resulted in a nearly linear increase in caloric content. Sodium chloride affected the lipid content of fourth instar larvae. The lipid content was higher with NaCl present in the media than without NaCl. Interaction had a significant effect on all responses except survival of third and fourth instar larvae. Phenol-C¹⁴ was metabolized by bacteria, but not by C. attenuatus.     

松墨天牛成虫雌雄体型差异及蛹和成虫大小与越冬幼虫体重的关系

【目的】本研究旨在明确松墨天牛 Monochamus alteratus 成虫及越冬幼虫体型大小的差异,探讨该虫越冬后成虫体型大小和越冬幼虫体重大小关系及原因。【方法】于2014年5月1日至10月31日在浙江富阳野外诱捕松墨天牛,通过测量野外诱捕到的松墨天牛成虫体长、体宽,确定林间松墨天牛成虫体型的差异;松墨天牛越冬幼虫的采集和称重测量明确越冬幼虫的组成和体重大小差异;进而通过对越冬幼虫单头跟踪饲养至化蛹、羽化,确定越冬幼虫体重大小与发育所得蛹和成虫大小的关系,阐明林间松墨天牛成虫体型差异的原因。【结果】浙江富阳野外诱捕发现,松墨天牛的活动期间很长,从5月中旬到10月初一直能诱捕到松墨天牛成虫,高峰期在6和7月份。松墨天牛雌雄成虫体型差异很大,雌虫平均体长和体宽分别为20.59±0.19和6.59±0.06 mm;雄虫平均体长和体宽分别为19.90±0.26 和6.44±0.08 mm; 雌虫的平均体长明显高于雄虫,但二者体宽没有显著差异;并且雌雄成虫体长和体宽呈显著正相关。越冬幼虫的头宽测定表明头宽的变化很大,最小为2.20 mm,最大为4.24 mm,经比对越冬幼虫由4龄和5龄幼虫组成;幼虫体重差异大,平均体重为304.2 mg, 介于71.6~858.0 mg之间,其中5龄越冬幼虫显著重于4龄越冬幼虫。进一步将越冬幼虫单管饲养跟踪研究发现,越冬幼虫体重大小决定蛹和羽化后的成虫的大小,二者存在显著的正相关,并且由4龄越冬幼虫发育所得的蛹和成虫显著轻于由5龄幼虫发育所得的蛹和成虫。【结论】松墨天牛成虫体型差异很大,这与越冬幼虫体重差异相关;越冬幼虫的体重大小决定了其化蛹后的蛹重和羽化后成虫的体型和体重的大小;造成越冬幼虫体重差异的可能原因包括松墨天牛成虫扬飞周期长导致的产卵期长而使得天牛发育进度不一致以及寄主不同部位营养的差异。     

Reciprocal transplantation of wing discs between a wing deficient mutant (fl) and wild type of the silkworm, Bombyx mori

The wingless mutant flügellos ( fl ) of the silkworm lacks all four wings. Although wing discs of the fl seem to develop normally until the fourth larval instar, wing morphogenesis stops after the fourth larval ecdysis, probably caused by aberrant expression of an unidentified factor, referred to as fl . To characterize factor fl , the wing discs dissected from the wild-type (WT) and fl larvae were transplanted into other larvae and developmental changes of the discs were examined. When the wing disc from a WT larva was transplanted into another WT larva and allowed to grow until emergence, a small wing appeared that was covered with scales. Thus, the transplanted wing discs can develop autonomously, form scales and evert from adult skin. The WT wing discs transplanted into the fl larvae also developed at a high rate. However, the fl wing discs transplanted into the WT larvae did not develop during the larval to pupal developmental stages. These data suggest that the fl gene product (factor fl) works in the wing disc cells during wing morphogenesis. Its function cannot be complemented by hemolymph in the WT larva. It is also implied that the level of humoral factors and hormones required for wing morphogenesis are normally maintained in the fl larva.     

Analysis of precocious metamorphosis induced by application of an imidazole derivative to early third instar larvae of the silkworm,Bombyx mori

When an imidazole derivative (KK-42) was applied to day 1 third instar larvae of the silkworm, Bombyx mori, 100% underwent precocious metamorphosis at the end of the fourth instar. Thus, the fourth instar becomes the last instar in these KK-42–treated larvae. The endocrine systems underlying the precocious metamorphosis were analyzed in the present study. Hydroprene application during the prolonged third instar after KK-42 treatment can prevent precocious metamorphosis, and the results showed dose-dependent and stage-specific effects. From analysis of the developmental changes in ecdysteroid levels in both KK-42–treated larvae and KK-42– and hydroprene-treated larvae, we conclude that changes in JH levels during the third larval instar can modify the secretion pattern of prothoracic glands and that during the next larval instar, very low ecdysteroid levels during the early stages of the presumptive last (fourth) larval instar are directly related to precocious metamorphosis. Arch. Insect Biochem. Physiol. 36:349–361, 1997. © 1997 Wiley-Liss, Inc.     

Effects of a juvenoid, DPE-28, on biology and behaviour of Culex quinquefasciatus, the human filariasis vector

Studies on the effect of a juvenoid, DPE-28 (2,4-dinitrophenyl-2',6'-di-tertiarybutyl phenyl ether) on biology and behaviour of Cx. quinquefasciatus showed that the developmental duration, sex ratio, mating success and blood feeding were considerably affected by the exposure of larvae and pupae to the compound. Exposure of fourth instar larvae to 0.007 (EI90) and 0.0019 (EI50) ppm of DPE-28 prolonged the duration of pupation by 58.6 and 52.4 hr and delayed the adult emergence by 35.4 and 17.7 hr in males and 36.8 and 21.1 hr in females respectively. Exposure of freshly ecdysed pupae to 10 and 5 ppm delayed the adult emergence with respect to the control by 54.3 and 32.4 hr in males and 55.2 and 33.2 hr in females respectively. The sex ratio of the adults emerged from treated larvae and pupae was also affected. The female mosquitoes that survived from the exposed fourth instar larvae and pupae exhibited a low blood engorgement ratio. This depression in blood feeding was more pronounced in adults emerged from treated pupae than that of treated fourth instar larvae. A significant proportion of adults emerged from treated larvae and pupae were able to feed only partially. Mating success of the treated populations declined considerably when crosses were made between the males and females emerged from treated fourth instar larvae and pupae. The adults emerged from treated larvae and pupae showed a significant reduction in the oviposition.     

Laboratory evaluation of certain natural compounds against the melon ladybird beetle,Epilachna chrysomelina F. attacking cucurbit plants

Neem Azal-T/S, Neudosan and Spruzit flüssig at different concentrations were evaluated against the second and fourth instar larvae of E. chrysomelina under laboratory conditions. In case of acute effects, the highest mortality was recorded when larvae were offered squash leaves treated with the highest concentration (2%) of Neem Azal-T/S, and the lowest mortality was, however recorded after treating them with the lowest concentration (0.25%). On the other hand, the second instar larvae were more susceptible to any compound at any concentration than the fourth instar one. One per cent Neudosan formulation showed the highest toxicity to both the 2nd and 4th instar larvae. As the concentration of this compound decreased the mortality percentage of treated larvae decreased to reach the minimum at the lowest concentration (0.25%). The same trend could be applied for Spruzit flüssig compound. When Neem Azal-T/S was compared with Neudosan, the later at any concentration was more toxic than the former. Generally, Neudosan and Spruzit flüssig seemed to be active against E. chrysomelina during the first three days after treatment, then, deterioration occurred among this compound. In case of latent effects, the duration of the treated fourth instar larvae of E. chrysomelina with any tested compound, was prolonged. An increase in the concentration caused prolongation of this period to reach the longest at the highest concentration. This prolongation, was more pronounced after Neem Azal-T/S treatment followed by Neudosan and Spruzit flüssig. The same trend could be applied for the duration of the pupal stage as being affected by the different concentrations of the tested compounds which were applied to the fourth instar larvae. However, adults of E. Chrysomelina treated as fourth instar larvae with high concentrations of Neem Azal-T/S showed some adult malformations. The sex ratios of adults produced from fourth instar larvae treated with different concentrations of the tested natural compounds were about 1?:?1, except for two cases; 1% Neudosan where the number of females were double the number of males and 0.5% Spruzit flüssig where the number of males were double the number of females. An increase in the concentration of any tested compound caused an obvious decrease in the percentage of emerged adults. The least emergence percentage was recorded in the case of Spruzit flüssig, followed by Neudosan and Neem Azal-T/S. Treating the fourth instar larvae with the different concentrations of the tested natural compounds significantly shortened the longevity of produced adults and decreased the fecundity.     

Allatotropic and allatostatic activities in extracts of brain and the effects of Manduca sexta allatotropin and Manduca sexta allatostatin on juvenile hormone synthesis in vitro by corpora allata from the Eri silkworm, Samia cynthia ricini

Both allatotropic and allatostatic activities were found in crude extracts of brain from adult and larval Eri silkworm, Samia cynthia ricini, but it seems that allatotropic activity dominates in each stage. There was a high level of allatotropic activity in the crude extract of brain from newly emerged female adults, but allatostatic activity appeared in the bioassay when excessive amounts of crude extracts of brain were added. Crude extracts of brain from premoulting fourth‐instar larvae and from newly ecdysed fifth‐instar larvae exhibited allatotropic activities, whereas extracts of brain from the second and third day of the fifth‐instar larvae inhibited juvenile hormone (JH) release slightly. Allatotropic activity from the brains of adults and larvae stimulated both adult and larval corpora allata (CA) to synthesize JH. Manduca sexta allatotropin (AT) (Mas‐AT) and M. sexta allatostatin (AST) (Mas‐AST) also stimulated and inhibited both adult and larval S. cynthia ricini CA to synthesize JH, respectively. Higher concentrations of Mas‐AT (10^?4 or 10^?3 M) showed an inhibitory effect on adult CA. CA from newly emerged female adults were the most sensitive to inhibition by Mas‐AST, whereas CA from female pharate adults at about 6 h before adult emergence were the most sensitive to stimulation by Mas‐AT and S. cynthia ricini brain allatotropic activity. An extract of brain and Mas‐AT induced some of the non‐active female pharate adult CA at 12 h before emergence to synthesize a small amount of JH.     

Developmental and sex-dependent regulation of storage protein synthesis in the silkworm,Bombyx mori

The mechanism of sex-dependent expression of a major plasma protein, referred to as storage protein 1 (SP-1) was studied during development of the silkworm, Bombyx mori. SP-1 occurred in the hemolymph of the female as well as in the male larvae until the end of the fourth larval instar. In the last instar larvae, the amount of SP-1 in the hemolymph greatly increased in females, but markedly declined in males. The level of fat body mRNA for SP-1 reflected the developmental and sex-dependent changes in the hemolymph concentration of SP-1. The developmental patterns of hemolymph proteins in the third and the fourth instar larvae of sex-mosaic individuals were quite analogous to those observed in normal larvae at the same developmental stages. The hemolymph concentration of SP-1 at the last larval instar of the sex mosaics varied among individuals irrespective of the gonad compositions. In vitro culture of the fat body cells dissected from several locations of a sex-mosaic larva provided evidence that each fat body cell in a common hemolymph milieu synthesizes a high (female type) or a low (male type) level of SP-1 depending on the sex chromosome composition. The amount of vitellogenin in the hemolymph of the sex-mosaic pupae was in proportion to that of SP-1 at the last larval instar. From these results, it is suggested that the sex-dependent expression of SP-1 and vitellogenin in B. mori is genetically determined and developmentally regulated without participation of the reproductive organs or any sex-specific humoral factors.     

马尾松毛虫过氧化氢酶及过氧化物酶与耐药性的关系

马尾松毛虫Dendrolimus punctatus幼虫体内存在着过氧化氢酶(CAT)和过氧化物酶(POD)。4龄幼虫的CAT和POD活力较大,其次是6龄幼虫,5龄幼虫的CAT和POD活力较4龄和6龄幼虫低。醚菊酯(etofenprox)处理后,在兴奋期(30 min),CAT和POD活力水平上升。4龄和6龄幼虫在抑制期(50 min以后),CAT和POD活力呈波动式上升,接近死亡时下降。5龄幼虫的CAT和POD活力呈波动式上升,接近死亡时下降。5龄幼虫的CAT在抑制期保持比正常虫体高的活力。结果表明,马尾松毛虫4龄、5龄和6龄幼虫与耐药性存在一定的相关性,研制酶的抑制剂具有实用意义。根据毒力测定结果,马尾松毛虫幼虫对醚菊酯的耐药力,5龄是4龄的1.43倍,6龄是4龄的1.72倍。因此,药物防治的合理时期应掌握在4龄以前较适宜。     

The management of metabolic energy storage during the life cycle of mayflies: a comparative field investigation of the collector-gatherer <Emphasis Type="Italic">Ephemera danica</Emphasis> and the scraper <Emphasis Type="Italic">Rhithrogena semicolorata</Emphasis>

The concentration and seasonal dynamics of the major energy storage components, triglycerides and glycogen, were measured in two species of mayfly (Rhithrogena semicolorata and Ephemera danica) with contrasting life cycle strategies living in a small mountain stream. E. danica is a burrowing, semivoltine collector-gatherer; R. semicolorata is univoltine and scrapes periphyton from stones. This is the first publication which focuses on the role of metabolic energy sources during the larval life span of two mayfly species until the larvae emerge. Although triglycerides are the major energy reserve in both species (>84% of total energy storage) throughout the whole larval development their seasonal dynamic differed considerably. In R. semicolorata the triglyceride concentration declined during the last weeks prior to emergence in both sexes. The same pattern was found in female larvae of E. danica, but not in male E. danica. It is suggested that females use triglycerides in the last larval stages for egg maturation, which is completed in the last larval instar. In male E. danica the triglyceride concentrations remained high until emergence, presumably due to their high energy demands as adults for their swarming flights. Glycogen concentrations did not show such a difference between species and sexes. Its significance as a storage substrate for energy is rather low; however, concentrations decreased in both species and sexes prior to emergence.     

Hemolymph titers of the biliprotein,insecticyanin, during development of Manduca sexta

The levels of a biliverdin-associated protein in the hemolymph of larvae, pupae and adults, and in egg homogenates, of Manduca sexta were determined by radial immunodiffusion. The concentration of the protein fluctuates dramatically during development and displays an ontogenetic pattern different from that of the total hemolymph protein concentration. During the larval stages studied, the very early fourth instar displayed the highest concentration of insecticyanin (0.6 mg/ml), which dropped precipitously afterwards (0.3 mg/ml). During fifth instar development, the levels decreased after ecdysis (0.15 mg/ml), began to rise at wandering, and nearly doubled (0.3 mg/ml) by the time of pupation. Pupal titers of the protein remained fairly constant until the day before adult eclosion, when titers increased. The highest levels of any stage were recorded for adults 12 hr post eclosion (0.80 mg/ml).     

Effects of Bt transgenic rice line on stripe stem borer,Chilo suppressalis and its consequences on egg parasitoid,Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) in the laboratory

Interactions among Chilo suppressalis (Walker), its larval parasitoid Trichogramma brassicae Bezdenko and insect-resistant transgenic rice lines were evaluated under laboratory conditions. The results showed that all neonates and second larvae of stem borer were dead regardless of being fed rotationally or permanently on Bt rice, but 18% and 28% of the third and fourth instar larvae could complete development and turned to adults, respectively, when fed rotationally. In the second year of project, third instar larvae of stem borer were used to evaluate Bt rice impact on T. brassicae. The result showed that emergence rate was lower, the abnormal female rate increased and adult female longevity of the wasp decreased significantly in comparison with the conventional variety. There was no significant difference in gender ratio and parasitism rate under Bt rice treatment in comparison with conventional variety.     

Laboratory evaluation of Alsystin, a chitin synthesis inhibitor, against Culex pipiens pipiens L. (Dip., Culicidae): effects on development and cuticle secretion

Alsystin, a benzoylphenylurea derivative, was evaluated on Culex pipiens pipiens L. Treatment was made on newly third- and fourth-instar larvae for 24 h. Mortality occurred at various developmental stages following treatment. However, death as larvae was relatively important compared with the mortality recorded for the other later developmental stages. Treatment resulted in a dose-dependent reduction in adult emergence. Moreover, treatment caused morphological aberrations depending upon the importance of larval–adult transformation and significantly increased the duration of both third and fourth larval instars. A histological study conducted on fourth instar larval integument, revealed that Alsystin delayed the ecdysis and significantly reduced the thickness of both larval and pupal cuticles secreted compared with controls. Moreover, the apolysis that occurred at day 4 in controls was not affected by treatment.     

Biology of Microplitis kewleyi (Hymenoptera: Braconidae): A parasite of Agrotis ipsilon (Lepidoptera: Noctuidae) larvae

Microplitis kewleyi Muesebeck is a gregarious internal parasite of larvae of the black cutworm Agrotis ipsilon (Hufnagel). Studies of the biology of the parasite revealed that there was an inverse relationship between host instar and parasite preference. Duration of development from egg to pupa ranged from 18 days at 27°C to 68.7 days at 16°C. Development from egg to pupa took 13.5–21.6 days when fourth and first instar host larvae, respectively, were parasitized. A larger number of parasites emerged from hosts parasitized in the fourth instar (22.4) than the first instar (11.5). Parasite pupation occurred when the host was in the fifth/sixth instar, depending on the instar parasitized. Thirty‐nine per cent of host larvae exposed as first instars to parasites died before parasite emergence. This decreased to 0% for host larvae exposed as fourth instars. The sex ratio was 1:1.2 (M:F). Thirty‐seven per cent of hosts exposed diurnally were stung, compared to 24% exposed nocturnally. Mean daily progeny was highest (12) on the first day, decreasing to zero after 20 days. Percent host parasitism was also highest on the first day (35%) decreasing to nearly 0% after 18 days. There appear to be three parasite larval instars. Host larvae often remained alive after parasite emergence.     

Olfactory learning and memory in Grapholita molesta

This study aimed to evaluate the effect of previous exposure to the essential oil of lemongrass [Cymbopogon citratus (DC) Stapf (Poaceae)] on the chemotactic behavior of Grapholita molesta (Busck) (Lepidoptera: Tortricidae) larvae and adults. All insect responses were evaluated using a Y‐tube (dual choice) olfactometer. Experience with the essential oil for 48 h after hatching did not change the chemotactic behavior of larvae; however, when they were exposed to lemongrass for 10–12 consecutive days, they did not present a preference for either treatment. Adults originating from larvae exposed to the essential oil odor until the formation of pupae responded more to this treatment in comparison to acetone. Similar results were obtained with insects exposed for only 24 h (at the end of the last instar) to the same treatment. Similar to inexperienced individuals, adults exposed for 48 h after emergence to acetone were significantly more responsive to this compound than to the essential oil. However, insects exposed to lemongrass odor during the same period displayed no preference for either treatment. This study demonstrated that both larvae and adults of G. molesta are capable of non‐associative learning and the memory acquired in the larval stage is maintained for at least 48 h after emergence.