THE EFFECTS OF XANTHOTOXIN ON THE BIOLOGY AND BIOCHEMISTRY OF Galleria mellonella L. (LEPIDOPTERA: PYRALIDAE)

The effects of a dietary plant allelochemical, xanthotoxin (XA), on survivorship, development, male and female adult longevity, fecundity, and hatchability of the greater wax moth Galleria mellonella L. were investigated. Oxidative stress indicators, the lipid peroxidation product, malondialdehyde (MDA), and protein oxidation products, protein carbonyl (PCO) contents, and activities of a detoxification enzyme glutathione S‐transferase (GST) activity were determined in wax moth adults. The insect was reared from first‐instar larvae on an artificial diets containing XA at 0.001, 0.005, or 0.1% to adult stage in laboratory conditions. Relative to the controls, the diets containing XA concentrations led to decreased survivorship in seventh instar, pupal, and adult stages. Compared to control diet (77.7%), the highest dietary XA concentration decreased survivorship to adulthood to 11.0%. The highest XA concentration (0.1%) reduced female longevity from 10.4 to 5.7 days and decreased egg numbers from 95.0 to 33.5 and hatchability from 82.7 to 35.6%. The lowest XA concentration (0.001%) led to about a sixfold increase in MDA content. XA at high concentrations (0.005 and 0.1%) increased MDA (by threefold) and protein carbonyl (by twofold) contents decreased GST activity. The highest dietary XA concentration decreased GST activity from 0.28 ± 0.025 to 0.16 ± 0.005 μmol/mg protein/min. We infer from these findings that XA‐induced oxidative stress led to decreased biological fitness.     

THE INFLUENCE OF DIETARY α‐SOLANINE ON THE WAXMOTH Galleria mellonella L

Plant allelochemicals are nonnutritional chemicals that interfere with the biology of herbivores. We posed the hypothesis that ingestion of a glycoalkaloid allelochemical, α‐solanine, impairs biological parameters of greater wax moths Galleria mellonella. To test this idea, we reared wax moths on artificial diets with 0.015, 0.15, or 1.5 mg/100 g diet of α‐solanine. Addition of α‐solanine to the diet affected survival of seventh‐instar larvae, pupae, and adults; and female fecundity and fertility. The diet containing the highest α‐solanine concentration led to decreased survivorship, fecundity, and fertility. The diets supplemented with α‐solanine led to increased malondialdehyde and protein carbonyl contents in midgut and fat body and the effect was dose‐dependent. Dietary α‐solanine led to increased midgut glutathione S‐transferase activity and to decreased fat body glutathione S‐transferase activitiy. We infer from these findings that α‐solanine influences life history parameters and antioxidative enzyme activities in the midgut and fat body of G. mellonella.     

The effects of boric acid-induced oxidative stress on antioxidant enzymes and survivorship in Galleria mellonella

Larvae of the wax moth, Galleria mellonella (L.), were reared from first instar on a diet supplemented with 156, 620, 1,250, or 2,500 ppm boric acid (BA). The content of malondialdehyde (MDA, an oxidative stress indicator), and activities of the antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione peroxidase (GPx)] were determined in the fat body and hemolymph in the 7th instar larvae and newly emerged pupae. Relative to control larvae, MDA was significantly increased in larval hemolymph, larval and pupal fat body, but decreased in the pupal hemolymph. Insects reared on diets with 156- and 620-ppm BA doses yielded increased SOD activity but 1,250- and 2,500-ppm doses resulted in decreased SOD activity in larval hemolymph. SOD activity was significantly increased but CAT was decreased in the larval fat body. High dietary BA treatments led to significantly decreased GST activity. However, they increased GPx activity in larval hemolymph. Dietary BA also affected larval survival. The 1,250- and 2,500-ppm concentrations led to significantly increased larval and pupal mortality and prolonged development. In contrast, the lowest BA concentration increased longevity and shortened development. We infer that BA toxicity is related, at least in part, to oxidative stress management.     

Boric acid-induced effects on protein profiles of Galleria mellonella hemolymph and fat body

The dietary effects of boric acid (BA) on the protein profiles of greater wax moth, Galleria mellonella (L.), were investigated in hemolymph and fat body of final instar (VIIth) and pupae. The insects were reared from first-instar larvae on an artificial diets containing 156, 620, 1250 or 2500 ppm of BA. We detected many undetermined protein fractions (6.5-260 kDa) in addition to well-defined protein fractions such as lipophorins and storage proteins in the tissues by using sodium dodecyl-sulphate polyacrylamide gradient gel electrophoresis. A marked quantitative change in the 45 kDa protein fraction of the hemolymph was observed in the VIIth instar larvae reared on 2500 ppm dietary BA.     

Malathion-induced oxidative stress in a parasitoid wasp: effect on adult emergence, longevity, fecundity, and oxidative and antioxidative response of Pimpla turionellae (Hymenoptera: Ichneumonidae)

Effects of an organophosphorus insecticide, malathion, on survivorship and lipid peroxidation of the greater wax moth, Galleria mellonella (L.), pupae were investigated by rearing the newly hatched larvae on an artificial diet containing 0.01, 0.1, 1, 10, and 100 ppm of the insecticide. As bioindicators of long-term physiological stress responses, the adult emergence rate, longevity, and fecundity associated with lipid peroxidation level and antioxidant enzyme activity in the endoparasitoid Pimpla turionellae (L.) (Hymenoptera: Ichneumonidae) were determined by rearing the parasitoid on a factitious host, G. mellonella pupae treated with malathion. At 100 ppm, malathion significantly decreased pupation rate of G. mellonella larvae and the rate of adult emergence of the parasitoid from these pupae. This concentration resulted in a significant increase in the lipid peroxidation product malondialdehyde (MDA) in both the host and the parasitoid. Malathion at 1 and 10 ppm significantly increased pupation rate and lipid peroxidation level of G. mellonella pupae. The adult emergence rate of P. turionellae was significantly decreased from 63.7 to 20% by these concentrations, whereas MDA content was increased by two- and three-fold, respectively, compared with the control (45.3 +/- 3.2 nmol/ g protein). The longevity of adults was significantly extended from 52.5 +/- 5.7 to 75.7 +/- 6.3 d when the parasitoids emerged from host pupae exposed with 0.1 ppm malathion. At low concentrations (0.01 and 0.1 ppm), malathion significantly increased the number of eggs laid per female per day. However, the lowest concentration (0.01 ppm) had no significant effect on hatchability, whereas 0.1 ppm of the insecticide resulted in significant decrease in egg hatch compared with the control. A significant increase in total superoxide dismutase (SOD) activity for low concentrations of malathion (0.01-1 ppm) was found compared with the control. There was a significant positive correlation of SOD activities with adult longevity and fecundity. This study suggested that malathion-induced oxidative stress was causative factor in the deterioration of biological fitness and that increased SOD activities may have resulted in decreased oxidative damage, which retarded the rate of deteriorative physiological changes in P. turionellae in response to sublethal doses of malathion.     

Penicillin-induced oxidative stress: effects on antioxidative response of midgut tissues in instars of Galleria mellonella

Penicillin and other antibiotics are routinely incorporated in insect culture media. Although culturing insects in the presence of antibiotics is a decades-old practice, antibiotics can exert deleterious influences on insects. In this article, we test the hypothesis that one of the effects of dietary penicillin is to increase oxidative stress on insects. The effects of penicillin on midgut concentrations of the oxidative stress indicator malondialdehyde (MDA) and on midgut antioxidant enzyme (superoxide dismutase [SOD], catalase [CAT], glutathione S-transferase [GST], and glutathione peroxidase [GPx]) and transaminases (alanine aminotransferase and aspartate aminotransferase) activities in greater wax moth, Galleria mellonella (L.), were investigated. The insects were reared from first instars on artificial diets containing 0.001, 0.01, 0.1, or 1.0 g penicillin per 100 g of diets. MDA content was significantly increased in the midgut tissues of each larval instar reared in the presence of high penicillin concentrations. Activities of antioxidant and transaminase enzymes did not show a consistent pattern with respect to penicillin concentrations in diet or age of larvae. Despite the increased penicillin-induced oxidative stress in gut tissue, antioxidant and transaminase enzymes did not correlate with oxidative stress level or between each other in larvae of other age stages except for the seventh instar. We found a significant negative correlation of MDA content with SOD and GST activities in seventh instars. SOD activity was also negatively correlated with CAT activity in seventh instars. These results suggest that exposure to dietary penicillin resulted in impaired enzymatic antioxidant defense capacity and metabolic functions in wax moth larval midgut tissues and that the resulting oxidative stress impacts midgut digestive physiology.     

Venturia canescens parasitizing Galleria mellonella and Anagasta kuehniella: differing suitability of two hosts with highly variable growth potential

Venturia canescens (Grav.) (Hymenoptera: Ichneumonidae) is a solitary larval koinobiont endoparasitoid, ovipositing in several larval instars of different pyralid moth species that are pests of stored food products. After oviposition, the host larva continues to feed and grow for at least several days, the precise time doing so depending on the stage attacked. We examined the relationship between host stage and body mass on parasitoid development in late second to fifth instars of two hosts with highly variable growth potential: the wax moth, Galleria mellonella (L) and the flour moth, Anagasta kuehniella (Zeller)(Lepidoptera: Pyralidae). G. mellonella is the largest known host of V. canescens, with healthy larvae occasionally exceeding 400mg at pupation, whereas those of A. kuehniella rarely exceed 40 mg at the same stage. Parasitoid survival was generally higher in early instars of G. mellonella than in later instars. By contrast, percentage adult emergence in A. kuehniella was highest in late fifth instar and lowest in late second instar. A. kuehniella was the more suitable host species, with over 45% adult emergence in all instars, whereas in G. mellonella we found less than 35% adult emergence in all instars. Adult parasitoid size increased and egg-to-adult development time decreased in a host size- and instar-specific manner from A. kuehniella. The relationship between host size and stage and these fitness correlates was less clear in G. mellonella. Although both host species were parasitized over a similar range of fresh weights, the suitability weight-range of A. kuehniella was considerably wider than G. mellonella for the successful development of V. canescens. However, in hosts of similar weight under 5 mg when parasitized, larger wasps emerged from G. mellonella than from A. kuehniella. Parasitoid growth and development is clearly affected by host species, and we argue that patterns of host utilization and resource acquisition by parasitoids have evolved in accordance with host growth potential and the nutritional requirements of the parasitoid.     

Eicosanoids mediate Galleria mellonella cellular immune response to viral infection

Nodulation is the predominant insect cellular immune response to bacterial and fungal infections and it can also be induced by some viral infections. Treating seventh instar larvae of greater wax moth Galleria mellonella with Bovine herpes simplex virus-1 (BHSV-1) induced nodulation reactions in a dose-dependent manner. Because eicosanoids mediate nodulation reactions to bacterial and fungal infection, we hypothesized that eicosanoids also mediate nodulation reactions to viral challenge. To test this idea, we injected G. mellonella larvae with indomethacin, a nonsteroidal anti-inflammatory drug immediately prior to intrahemocoelic injection of BHSV-1. Relative to vehicle-treated controls, indomethacin-treated larvae produced significantly reduced numbers of nodules following viral infection (down from approximately 190 nodules/larva to <50 nodules/larva). In addition to injection treatments, increasing dietary indomethacin dosages (from 0.01% to 1%) were associated with decreasing nodulation (by 10-fold) and phenoloxidase activity (by 3-fold) reactions to BHSV-1 injection. We infer from these findings that cyclooxygenase products, prostaglandins, mediate nodulation response to viral infection in G. mellonella.     

The involvement of protein kinase A in the immune response of Galleria mellonella larvae to bacteria

The role of protein kinase A (PKA) in the humoral immune response of the greater wax moth Galleria mellonella larvae to live gram-positive bacteria Micrococcus lysodeikticus and gram-negative bacteria Escherichia coli was investigated. The immune challenge of larvae with both kinds of bacteria caused an increase in fat body PKA activity depending on the injected bacteria. Gram-positive M. lysodeikticus was a much better inducer of the enzyme activity than gram-negative E. coli. The PKA activity was increased about 2.5-fold and 1.5-fold, after M. lysodeikticus and E. coli injection, respectively. The in vivo inhibition of the enzyme activity by a cell permeable selective PKA inhibitor, Rp-8-Br-cAMPS, was correlated with considerable changes of fat body lysozyme content and hemolymph antimicrobial activity in bacteria-challenged insects. The kinetics of changes were different and dependent on the bacteria used for the immune challenge of G. mellonella larvae.     

Presence of IL-1- and TNF-like molecules in Galleria mellonella (Lepidoptera) haemocytes and in an insect cell line Fromestigmene acraea (Lepidoptera).

In this study the authors give immunocytochemical evidence for the presence of interleukin (IL)-1alpha- and tumour necrosis factor (TNF) alpha-like molecules in the haemocytes of last instar larvae from the greater wax moth Galleria mellonella. Similar results are demonstrated in a continuous haemocyte line (BTI-EA-1174-A) from the salt marsh caterpillar Estigmene acraea. In Galleria mellonella larvae granular cells show a strong positive reaction with both primary antibodies, whereas plasmatocytes are stained to a lesser extent. Cell line haemocytes also react positively with both antibodies. After activating the cells with lipopolysaccharide (LPS) staining of Estigmene acraea cells is decreased, whereas Galleria mellonella haemocytes show no visible reaction in comparison to non-activated cells.     

A semi-synthetic diet for rearing Dipha aphidivora (Lepidoptera: Pyralidae), a promising predator of woolly aphid in sugarcane

Dipha aphidivora (Meyrick), a lepidopteran predator of sugarcane woolly aphid (SWA) Ceratovacuna lanigera Zehntner was successfully reared for three successive generations, for the first time on a freeze-dried beef liver-based larval semi-synthetic diet. We compared biological parameters viz., larval survival, adult weight, pre-and oviposition period, fertility, fecundity and female longevity of D. aphidivora reared on the semi-synthetic diet with the predators reared on SWA. Development time of larvae reared from first instar to pupation was 20.6 days on the semi-synthetic diet and 12 days on SWA, while survival of the larvae to adults was 61.8 and 91.8% on larval semi-synthetic diet and SWA, respectively. Fecundity recorded from semi-synthetic diet (41 eggs/female) was significantly less than those produced on SWA (58 eggs/female). However, fertility and longevity of the predators reared on SWA and semi-synthetic diet did not differ significantly. The study revealed the possibility of rearing D. aphidivora larvae using synthetic diet.     

Decanal as a major component of larval aggregation pheromone of the greater wax moth,Galleria mellonella

Larvae of the greater wax moth (GWM), Galleria mellonella, a destructive pest of the honeybee (Apis mellifera), have been observed to display aggregation behaviours. However, the underlying mechanism by which these larvae come together remains unknown. We hypothesized that the GWM larvae detect, orient towards and utilize conspecific larval chemical cues to aggregate in groups. We used dual‐choice olfactometer assays to investigate the involvement of conspecific larval odours in their aggregation amongst 3–5th instar and 8th instar larvae. The assays revealed that only 8th instar larvae were significantly attracted to their odours and those emanating from newly spun cocoons. Coupled gas chromatography–mass spectrometry (GC‐MS) of larval head space odours analysis revealed the presence of four compounds: nonanal, decanal, tridecane and tetradecane in pupal and mature larval odour extracts. However, using synthetic compounds, behavioural assays showed that only decanal induced significant attraction, therefore, suggesting its role as a major component of the larval aggregation pheromone of GWM. Our findings reveal the involvement of volatile organic compounds in the aggregation behaviour of mature wax moth larvae and thereby offer prospects for the development of an odour‐baited in‐hive trapping management tool for wax moth larva.     

Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria

Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.     

Reprogramming and DNA Synthesis in Galleria mellonella Larval Epidermal Cells

Epidermal cells from ligated day-3 last instar larvae of Galleria mellonella secrete a larval cuticle if exposed to ecdysone immediately following ligation; whereas, they deposit a pupal cuticle if exposed to ecdysone three to four days after ligation. During the period of reprogramming the genome in the diploid cells of the wax moth larvae no DNA synthesis occurs. This is direct proof that DNA synthesis is not required for reprogramming in the epidermis of Galleria during larval-pupal transformation.     

孟氏隐唇瓢虫成虫自残幼虫的研究

在室内对孟氏隐唇瓢虫成虫自残幼虫的行为以及雌虫取食幼虫后的生殖力进行了研究,结果表明,雌成虫在48h内残杀幼虫造成的死亡率为60%,显著高于雄成虫;与其他部位相比幼虫的腹部遭受更多地攻击,幼虫体表覆盖的蜡丝在一定程度上抵御了成虫的攻击。与取食粉蚧的雌虫相比,取食瓢虫幼虫的雌虫产卵率和平均产卵量均显著降低。     

Laboratory Effects of Ingestion of Azadirachtin by Two Pests ( Ceratitis capitata and Spodoptera exigua ) and Three Natural Enemies ( Chrysoperla carnea , Opius concolor and Podisus maculiventris )

The effects of azadirachtin on two pests: neonate larvae and newly emerged adults of Ceratitis capitata (Wiedemann) and last instar larvae of Spodoptera exigua (Hubner); and three natural enemies: newly emerged adults of Opius concolor Szepligeti, second instar larvae of Chrysoperla carnea (Stephens), and fifth instar nymphs of Podisus maculiventris (Say) were studied in laboratory. Adult insects were exposed to a non-oil formulation of azadirachtin (Align, emulsifiable concentrate, 3.2% azadirachtin, Sipcam Inagra, Spain) via their drinking water and immature instars were reared in the presence of the insecticide-treated diet. The natural enemies were exposed to at least the maximum field recommended concentration of the insecticide (0.15% v/v). Azadirachtin was highly toxic to neonate larvae of C. capitata and prevented adult emergence at a concentration of 1 mg a.i. l -1 . When adults were fed the insecticide at the maximum recommended concentration, their survival was not affected but egg laying was totally inhibited. Last instar S. exigua larvae were also very susceptible (LC 50 = 7.7 mg a.i. l -1 ) and at a concentration of 10 mg a.i. l -1 fecundity of surviving adults, and egg fertility, were reduced by 72 and 85%, respectively. Effects on O. concolor were large, and significant reductions in longevity, percentage of attacked hosts, and progeny size per female, were recorded. The predator P. maculiventris was much less sensitive to azadirachtin, but slight reductions in survival of emerged adults and of reproductive parameters occurred. The insecticide had no significant effect on C. carnea larvae fed with treated Sitotroga cerealella (Oliver) eggs, probably because of its inability to penetrate inside the egg.     

Temperature-dependent regulatory mechanism of larval development of the wax moth (Galleria mellonella)

The mechanisms underlying larval diapause in the wax moth (Galleria mellonella) is one of the most throughly studied aspects. At the low temperature of 18 degrees C, the last instar larvae did not pupate but transferred to 30 degrees C they initiated development and pupation in a circadian manner. Different types of surgical manipulations including head-ligation, nerve cord-severance, implantation of the brain, prothoracic glands, accompanied with ecdysteroid titre measurements indicated that diapausing arrest of larval development at 18 degrees C might be due to the nervous inhibition of their prothoracic glands.     

Dietary complementation across life stages in the polyphagous lady beetle Coleomegilla maculata

We investigated the life history consequences of changes in diet between larval and adult life stages in the polyphagous lady beetle Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae). Beetles were reared on three larval diets: greenbug, Schizaphis graminum Rondani (Homoptera: Aphididae), eggs of the flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae), and bee pollen. The reproductive performance of females was then evaluated on an adult diet of either greenbug or moth eggs. Moth eggs appeared to be the most suitable diet for larvae, yielding the largest adults, and pollen the least suitable, resulting in the smallest adults and greatly extended developmental time. Pollen‐reared beetles tended to have lower fecundity and fertility than those reared on animal protein, regardless of adult diet. Female fitness was generally increased by a change in diet upon emergence to the alternative source of animal protein, suggesting that dietary complementation occurred across life stages. Among females reared on greenbug, a change of diet to moth eggs reduced the period required for production of 12 clutches and increased egg fertility compared to continued feeding on greenbug. Among females reared on moth eggs, a change of diet to greenbug increased fecundity compared to continued feeding on moth eggs. Among females fed an adult diet of greenbug, those fed moth eggs as larvae had faster production of 12 clutches and higher fecundity. We discuss these novel results in the context of coccinellid life history and ecology and their potential implications for other insects that are predatory as both larvae and adults.     

Recognition and regulation of metalloproteinase activity in the haemolymph of Galleria mellonella: a new pathway mediating induction of humoral immune responses

Proteolytic activity released within an organism by wounded tissues or invading pathogens can strongly impair the physiological homeostasis when it remains non-regulated. Thus, an efficient mechanism that enables recognition and inactivation of non-regulated proteolytic activity is essential to limit toxic effects. In larvae of the Greater wax moth Galleria mellonella we discovered that injection of bacterial thermolysin at a sublethal concentration mediates both acquired resistance against a subsequently injected lethal concentration of this metalloproteinase and stimulation of humoral immune response accompanied by the synthesis of an inducible metalloproteinase inhibitor (IMPI) which is released within the haemolymph. In search of a putative mechanism mediating recognition and regulation of released microbial metalloproteinases we determined that thermolysin-mediated hydrolysis of G. mellonella haemolymph proteins in vitro yields small (<3 kDa), heat-stable molecules which were discovered to represent potent elicitors of humoral immune responses when injected into untreated larvae. Obtained results allowed to design a model explaining for the first time regulation of released metalloproteinases within the haemolymph of insects. The determined coherence between regulation of released metalloproteinases by IMPI and the simultaneous induction of antimicrobial proteins provides a new insight into the mechanisms leading to expression of genes in course of humoral immune responses.     

Assessment of sublethal effects of methoxyfenozide on oriental fruit Moth (Lepidoptera: Tortricidae)

Sublethal effects of the insect growth regulator methoxyfenozide were examined in oriental fruit moth, Grapholita molesta (Busck), in laboratory and field studies. In laboratory studies, oriental fruit moth larvae reared on diet amended with 0.1 ppm methoxyfenozide developed at the same rate as larvae reared on untreated diet, and paired moths reared as larvae from the same treated or untreated diets exhibited similar fecundity and fertility. Population growth differences over multiple generations were used to examine sublethal effects of methoxyfenozide on population dynamics in the field. Multiple single-tree cages were placed over apple (Malus spp.) trees treated with two applications of methoxyfenozide (70 g [AI] /ha) and nontreated trees. Cages were infested at a single time point with virgin male and female oriental fruit moth adults, and population growth was evaluated by egg counts, shoot infestation, fruit damage, and larval counts over a 12-wk period. Significantly fewer eggs, larvae, and damaged fruit were found on methoxyfenozide-treated compared with nontreated trees in 2001. Observed population differences may have been a result of direct mortality to eggs and larvae of the first generation rather than sublethal effects. In 2002, no differences were observed between treatments, but a heavy rain event shortly after the early infestation impacted the experiment. A late moth release treatment was tested in 2002 to examine the effects of residual methoxyfenozide 55 d after initial application. Significantly fewer eggs were found in the methoxyfenozide treatment compared with the control, but no differences existed among treatments in shoot infestation, percentage of damaged fruit, or larval populations. It was concluded direct mortality of eggs and larvae exposed to methoxyfenozide rather than sublethal effects were most important in reduction of subsequent generations.