Effect of 3 insect growth regulators on larval development,fecundity and egg viability of the coccinelidChilocorus bipustulatus [Col.: Coccinelidae]

The effect of 3 insect growth regulators — methoprene, diflubenzuron and RO 13-5223, on the coccinelidChilocorus bipustulatus L. was studied in the laboratory. Feeding onChrysomphalus aonidum (L.) orAspidiotus hederae Vallot (Diaspididae) treated with the IGRs at the concentration of 0.025% a.i. revealed the following: diflubenzuron caused a complete mortality of 1 st instar larvae; methoprene and RO 13-5223 did not arrest larval development but inhibited pupation; fecundity of sexually mature females was not affected by the 3 IGRs but egg hatch was completely inhibited; egg viability was regained when IGR-exposed females had been transferred to an uncontaminated environment.     

Studies on l-glutamate in insect haemolymph.

ABSTRACT. l -Glutamate when injected into the haemolymph of Lucilia sericata larvae and adult male Locusta migratoria was rapidly removed by uptake mechanisms to other tissues in the insect. Data from Lucilia larvae indicate that following uptake glutamate is metabolized and the metabolites are secreted back into the haemolymph. l -Aspartate injected into the haemolymph of Lucilia larvae was also rapidly removed. When both l -aspartate and l -glutamate were injected simultaneously, the rate of glutamate removal was significantly reduced. It is concluded that glutamate and aspartate share the same uptake mechanisms. l -Leucine injected into Lucilia larvae and Locusta was removed at a significantly slower rate than glutamate or aspartate.     

Morphological effects of insect growth regulating compounds on Aedes aegypti (Diptera: Culicidae) larvae.

Cuticular development of $\text{Aedes aegypti}$ larvae was examined by electron microscopy and comparisons were made between larvae exposed to methoprene, isopropyl (E, E)-11-methoxy-3, 7, 11-trimethyl-2, 4-dodecadienoate, those treated with the fluorescent insect growth regulator, 5-[[[5-(dimethylamino)-1-naphthalenyl]-sulfonyl]amino]-1, 3-benzodioxole (DNSAB), and untreated larvae. Larvae of all three groups were routinely fixed at 24, 48, and 72 hr posttreatment. Thin sections of the sixth-abdominal segment, anal papillae, midgut tissue, and Malpighian tubules were examined for morphological variations from controls.     

Morphological abnormalities and lethality in silkworm (Bombyx mori) larvae treated with high concentrations of insect growth-blocking peptide

Insect growth-blocking peptides (GBPs) exhibit growth-blocking and paralytic activity. Low concentrations of GBP stimulate larval growth, whereas high concentrations of GBP significantly retard larval growth. Here, we show that morphological abnormalities and lethality were induced in silkworm (Bombyx mori) larvae by high concentrations of GBP. Active B. mori GBP (BmGBP) was produced by treating recombinant proBmGBP (expressed in baculovirus-infected insect cells) with bovine factor Xa. When silkworm larvae on day 1 of the fifth-instar stage were injected between the seventh and eight abdominal segments with BmGBP (100 or 500 ng/larva), the larval–pupal and pupal–adult transformations of these silkworms were delayed in a dose-dependent manner. However, a high concentration (2000 ng/larva) of BmGBP or Spodoptera exigua GBP (SeGBP) acutely induced morphological abnormalities and death in silkworm larvae. In silkworm larvae treated with high concentrations of GBPs, the ingested food excessively accumulated in the foregut, which caused extreme swelling in both the thorax and the foregut and resulted in larval death. Therefore, these results not only provide insight into the effect of insect GBPs on gut physiology but also reveal a novel function of insect GBPs.     

An alternative insect pathogenic strategy in an Aspergillus flavus auxotroph

In order to study fungal pathogen evolution, we used a model system whereby the opportunistic fungus Aspergillus flavus was serially propagated through the insect (Galleria mellonella) larvae, yielding a cysteine/methionine auxotroph of A. flavus with properties of an obligate insect pathogen. The auxotroph exhibited insect host restriction but did not show any difference in virulence when compared with the wild-type (Scully LR, Bidochka MJ, 2006. Microbiology 152, 223-232). Here, we report that on 1 % insect cuticle medium and synthetic Galleria medium, the auxotroph displayed increased extracellular protease production, a virulence factor necessary for insect pathogenesis. In the wild-type strain, protease production was deregulated during carbon (glucose), nitrogen (nitrate), or sulphate deprivation. If all three were present, protease production was vastly reduced. However, in the cysteine/methionine auxotroph, protease production was deregulated in complete medium. We suggest that the deficiency in sulphate assimilation in the auxotroph resulted in deregulation of protease production. The auxotroph exhibited delayed germination and slower hyphal growth when compared to the wild-type but there were no differences in virulence or cuticle penetration, suggesting a shift in pathogenic strategy that compensated decreased growth with increased virulence factor (extracellular protease) production. We concluded that the biosynthetic deficiency that mediated insect host restriction also increased protease production in the slow-growing auxotroph, resulting in an alternate, more host-specific pathogenic strategy. However, we argue that transmission is not necessarily correlated with virulence as competition bioassays in insect larvae showed that the wild-type generally out-competed the auxotroph by producing the majority of the conidia on the sporulating cadavers. This is one of the few examples that highlight the effect of genome decay on nutrition acquisition, virulence, and transmission in fungal pathogen evolution.     

Persistence of the insect growth regulator Dimilin® in brackish water: a laboratory evaluation using larvae of an estuarine crab as indicator

The persistence of Dimilin® (diflubenzuron), an insect growth regulator which interferes with chitin formation in the cuticle of insect larvae, has been studied using larvae of the estuarine brachyuran crabRhithropanopeus harrisii (Gould) as test material. The results of the present investigation show that Dimilin breaks down relatively slowly in brackish water. It took about 8 weeks before a 10 ppb solution of Dimilin degraded to a level which did not affect survival of the crab larvae. Earlier it was shown (Christiansen et al., 1978) that nearly 100% ofR. harrisii larvae at each of the four zoeal stages died when molting to the succeeding stage after only 3 days of exposure to 10 ppb Dimilin. Hence, one should be extremely cautious in using Dimilin in estuarine areas where crab larvae occur.     

Insect resistance of transgenic tobacco expressing an insect chitinase gene

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control     

A novel secreted protein toxin from the insect pathogenic bacterium Xenorhabdus nematophila

The bacterium Xenorhabdus nematophila is an insect pathogen that produces several proteins that enable it to kill insects. Screening of a cosmid library constructed from X. nematophila strain A24 identified a gene that encoded a novel protein that was toxic to insects. The 42-kDa protein encoded by the toxin gene was expressed and purified from a recombinant system, and was shown to kill the larvae of insects such as Galleria mellonella and Helicoverpa armigera when injected at doses of around 30-40 ng/g larvae. Sequencing and bioinformatic analysis suggested that the toxin was a novel protein, and that it was likely to be part of a genomic island involved in pathogenicity. When the native bacteria were grown under laboratory conditions, a soluble form of the 42-kDa toxin was secreted only by bacteria in the phase II state. Preliminary histological analysis of larvae injected with recombinant protein suggested that the toxin primarily acted on the midgut of the insect. Finally, some of the common strategies used by the bacterial pathogens of insects, animals, and plants are discussed.     

TOXICITY AND GROWTH-REGULATING ACTIVITY OF A NEEM SEED KERNEL EXTRACT (AZAL-S) TO THE LARVAE OF CULEX QUINQUEFASCIATUS

Abstract Toxicity of a neem seed kernel extract (AZAL-S) was assessed under laboratory conditions against larvae of Culex quinquefasciatus . The LC₅₀ value for AZAL-S was 0.78× 10–6. Exposure of 4th instar larvae to 0. 3× 10–6 and 0. 5× 10–6 AZAL-S for 120 hours caused 46. 2% and 67. 5% mortality respectively, with various types of morphogenetic aberration. AZAL-S induced an obvious prologation of the larval period when 1st instar larvae were treated with 0. 4–1. 0× 10–6 AZAL-S for 7 days. The insect growth regulating effect of AZAL-S on mosquito larvae was similar to those reported for certain insect growth regulators.     

Effects of two insect growth regulators and a neonicotinoid on various life stages of <Emphasis Type="Italic">Aphytis melinus</Emphasis> (Hymenoptera: Aphelinidae)

The effect of two insect growth regulators and a neonicotinoid insecticide were tested on immature stages and adults of the parasitoid Aphtyis melinus DeBach (Hymenoptera: Aphelinidae), a key natural enemy of California red scale, Aonidiella aurantii (Maskell) (Hemiptera: Diaspididae), in California. No significant effects of the insect growth regulators on survival or development to the adult stage were found when the parasitoid was treated at any of the egg, larval, or pupal stages. The broad-spectrum neonicotinoid acetamiprid also showed no significant effect on the development of A. melinus to the pupal stage, probably because immature stages of this ectoparasitoid are protected under the cover of its armored scale host. However, 48 h exposure of adults to acetamiprid residues following emergence resulted in high levels of wasp mortality. Aphytis melinus adults treated with either of the two insect growth regulators as larvae survived 48 h exposure to pesticide residues as adults and showed levels of fecundity comparable with control insects. We conclude that the two insect growth regulators are compatible with augmentative releases of A. melinus but that treatments of acetamiprid should be avoided in situations where biological control by this parasitoid is important.     

Indirect plant-mediated effects on insect immunity and disease resistance in a tritrophic system

Host plant quality can significantly influence the growth and condition of phytophagous insects, and consequently their susceptibility to pathogens. This study examined the relationship between host plant quality, insect condition, immune responsiveness and resistance to pathogens in the cabbage looper, Trichoplusia ni. Two baseline and induced immune parameters were estimated, haemocyte numbers and haemolymph phenoloxidase (PO) activity, for larvae on two host plants, broccoli and cucumber. Haemolymph protein concentration was assessed as an indication of insect condition, and the susceptibility of larvae to T. ni single nucleopolyhedrovirus (SNPV) was used as a measure of disease resistance. T. ni growth, survival and condition was much higher on broccoli than cucumber. Haemocyte numbers were significantly higher in broccoli-reared larvae, whereas PO activity was not. An immune challenge induced significantly elevated numbers of haemocytes for larvae reared on both host plants, but did not affect PO activity or protein concentrations. Susceptibility to T. ni SNPV was markedly higher in larvae reared on cucumber than on broccoli. These results clearly indicate that host plant quality can affect both immune response and disease resistance of T. ni larvae and that bottom-up effects could be important in interactions between insects and entomopathogens.     

Storage and mobilisation of nutriment for uterine milk synthesis by Glossina morsitans

Nitrogen content of eggs and larvae of Glossina morsitans was a constant proportion of dry weight and equivalent to ca. 55% protein assuming tsetse proteins contain 16% nitrogen. The larval gut content (uterine milk) contained 40% protein. Fatty acid composition of lipids in the milk and in the larval body was similar, with Palmitic (35–38%), Palmitoleic (31–35%) and Oleic acid (23–25%) predominating. Results support the hypothesis that uterine milk contains both protein and lipid and that its composition is relatively constant throughout the period of its synthesis and secretion.Patterns of incorporation of radioactivity by fertilized adult females from injected [¹⁴C]-leucine changed throughout a pregnancy cycle. High levels of incorporation into lipid (22–30%) during early pregnancy fell to around 10% during late pregnancy. Over the same period low levels of incorporation into protein (5%) increased to 15%. Results support the hypothesis that uterine milk is synthesized from a lipid store laid down in early pregnancy coupled with protein derived largely from blood meals ingested later. Such a system would not require the insect to store proteins for larval growth and is economical in terms of energy expenditure.     

Fatty acid synthetase complex from the insect Ceratitis capitata.

Fatty acid synthesis capacity of the insect Ceratitis capitata has been investigated in vitro from [1-14C]acetyl-CoA using homogenates at different stages of development. A maximum activity was observed after 5--6 days of larval development. But homogenates of the pharate adult insect did not show synthetic capacity of fatty acids. Fatty acid synthetase complex has been isolated from the particle-free supernatant fraction of homogenates from the 6-day C. capitata larvae. The enzyme complex was purified 182-fold with respect to the protein contained in the crude extract. The complex was homogeneous when analysed by gel filtration and by polyacrylamide-gel electrophoresis. The molecular weight was 5.2X10(5). The enzyme was dissociated into half-molecular subunits. Amino acid analysis, general properties, stability and kinetic constants (V and Km) for the substrates are reported. The fatty acid synthetase complex from the insect contains 42+/-1-SH residues and one phosphopatetheine moiety per 5.2X10(5). Activity was dependent on the presence of NADPH; FMN strongly inhibited the enzyme activity promoted by NADPH. The enzyme complex synthesized a range of fatty acid (10:0--18:0), palmitate being the predominant end product. The proportions of fatty acids synthesized varied with substrate concentrations. Fatty acids released from the complex were almost completely in the free form.     

Effects of some insect growth regulators on natural enemies of scale insects (Hom.: Coccoidea)

We report and discuss effects of four insect growth regulators: buprofezin, fenoxycarb pyriproxyfen and chlorfluazuron, at concentrations recommended for agricultural use on six species of natural enemies of homopteran pests. Dipping in buprofezin had no appreciable effect on adult mortality, oviposition and development ofComperiella bifasciata (Howard), (Hymenoptera: Encyrtidae). When exposed to hosts treated with buprofezin, percentage mortality of adultEncyrtus infelix Embleton (Encyrtidae) was low; buprofezin had some detrimental effect on immature stages ofE. infelix when applied prior to parasitization, but not when introduced after parasitization. Buprofezin had a slight effect on the immature stages ofCryptochaetum iceryae Williston (Diptera: Cryptochaetidae), while fenoxycarb and pyriproxyfen had marked detrimental effects on parasitization and/or development of the parasitoid fly. None of the larvae ofRodolia cardinalis Mulsant (Coleoptera: Coccinellidae) developed into adults after application of buprofezin, fenoxycarb or pyriproxyfen. Buprofezin and chlorfluazuron completely prevented egg hatch ofChilocorus bipustulatus L. (Coleoptera: Coccinellidae). Buprofezin did not adversely affect egg hatch and larval development ofElatophilus hebraicus Pericart (Hemiptera: Anthocoridae); fenoxycarb or pyriproxyfen applied either before or after oviposition on pine needles caused total suppression of egg hatch.     

Intrinsic glycosylation potentials of insect cell cultures and insect larvae

Summary The glycosylation and subsequent processing of native and recombinant glycoproteins expressed in established insect cell lines and insect larvae were compared. TheSpodoptera frugiperda (Sf21) andTrichoplusia ni (TN-368 and BTI-Tn-5B1-4) cell lines possessed several intrinsic glycoproteins that are modified with both N- and O-linked oligosaccharides. The N-linked oligosaccharides were identified as both the simple (high mannose) and complex (containing sialic acid) types. Similarly, theT. ni larvae also possessed intrinsic glycoproteins that were modified with O-linked and simple and complex N-linked oligosaccharides. Additionally, human placental, secreted alkaline phosphatase (SEAP) produced during replication of a recombinant baculovirus inT. ni larvae was modified with complex oligosaccharide having sialic acid linked α(2–6) to galactose.     

槲皮素对黄粉虫血淋巴酚氧化酶的生理效应

用酶标仪法测定了槲皮素对黄粉虫Tenebrio molitor酚氧化酶（phenoloxidase, PO）的生物活性。结果表明：在离体条件下,对黄粉虫血淋巴PO的IC₅₀为0.625 mmol/L。在活体情况下,当槲皮素-DMSO溶液或槲皮素水悬浮液（5 μL）注入虫体后,在浓度低于1.0 mmol/L时对黄粉虫血淋巴PO具有激活作用,当浓度高于2.0 mmol/L时则对PO具有明显的抑制作用;单独注射5 μL DMSO溶液对PO活性亦有一定的激活作用。试虫被注射槲皮素后,PO活性在2～4 h内迅速下降,然后缓慢上升,处理8 h左右达到最高点,其后低浓度处理PO活性保持不变,而高浓度处理则逐渐下降,提示低浓度槲皮素可以引起试虫的免疫反应。在PO测活体系中加入0.5％的BSA后对PO活性无影响,并能使槲皮素在测活体系中保持稳定。     

Immunosuppressive effect of cyclosporin A on insect humoral immune response

Cyclosporin A suppressed humoral immune response of Galleria mellonella larvae. Insects were immunized with LPS Pseudomonas aeruginosa and then injected with cyclosporin A. Immunosuppressive effects were expressed both, in larvae treated with cyclosporin A at the initial phase of immune response and at the effector phase of antibacterial immunity. Cyclosporin A moderately decreased lysozyme activity and significantly decreased antibacterial activity peptides against Escherichia coli. Immunosuppressive effects of cyclosporin A were observed after immunoblotting with antibodies anti-G. mellonella lysozyme. Tricine SDS/PAGE shown that synthesis of antibacterial peptides of larvae treated with cyclosporin A was considerably inhibited. Insects of impaired immune response by cyclosporin A action lost protective immunity to insect bacterial pathogen P. aeruginosa.     

Prey stage preference and feeding behaviour ofOligota kashmirica benefica (Coleoptera: Staphylinidae), an insect predator of the spider miteTetranychus urticae (Acari: Tetranychidae)

We studied the prey stage preference and feeding behaviour of the first to third instar larvae and adult females ofOligota kashmirica benefica Naomi (Coleoptera: Staphylinidae), a predator of the spider miteTetranychus urticae Koch (red form) (Acari: Tetranychidae), on leaves of the kudzu vine (Pueraria lobata (Wild.) Ohwi (Leguminosae)) under laboratory conditions. The number of mites eaten increased with the growth of predator larvae. Third instar larvae preyed on all stages of spider mite, whereas first instar larvae preyed mainly on immobile stages (eggs and quiescent stages). The predator larvae showed two types of foraging behaviour (active searching and ambush behaviour) when targeting the mobile stages (larval nymph and adult stages of prey). Although no difference was found in the number of prey consumed by adult females and third instar larvae of the predator, the adult females mainly attacked and consumed the immobile stages.     

Biological control of an insect pest by gut-colonizing Enterobacter cloacae transformed with ice nucleation gene

The ice nucleation (IN) gene inaA of epiphytic Erwinia (Pantoea) ananas IN10 was transformed into Enterobacter cloacae WBMH-3-CMr originated from the faeces of silkworms. The transformant designated as Ent. cloacae WBMH-3-CMr(pICE6S13) exhibited IN activity, unlike the parent strain. The transgenic strain was ingested by mulberry pyralid larvae, fed on detached mulberry leaves, and the supercooling capacity and cold hardiness of these larvae were examined. The mean supercooling point (SCP) of the larvae ingesting the transgenic strain was - 3.3 degrees C, 8 degrees C higher than that of larvae treated with distilled water (control) and 1.5 C higher than an ice nucleation active (INA) strain of Erw. ananas. The SCPs of the larvae were stably maintained over the 9 d after ingestion. The maintenance of these high SCPs was due to transgenic Ent. cloacae having a more stable and efficient gut colonization than Erw. ananas, which is identified by the distribution of a narrower range of SCPs (-2 to -5 degrees C) in larvae treated with the transgenic stain. Furthermore, most of the larvae ingesting the transgenic strain froze and died when they were exposed to cold conditions of -5 degrees C for 18 h, 3 or 7 d after ingestion. In contrast, most of the larvae ingesting no bacterium did not die under similar conditions. On the other hand, the growth ability of Ent. cloacae WBMH-3-CMr on mulberry leaves tended to be lower than that of epiphytic Erw. ananas, as assayed by pot tests. These findings would expand the possibility of biological control using INA bacteria since Ent. cloacae would harbour a broader host (insect) range for gut colonization and a smaller affinity to plants to benefit from prevention of plant frost injury.