Parasitism of the mosquito Culex pipiens by the nematode Heterorhabditis bacteriophora

Various concentrations of the nematode Heterorhabditis bacteriophora were added to dishes containing second, third, and fourth larval instars of the mosquito, Culex pipiens, respectively. The infective stage nematodes were ingested by the mosquito larvae, they then penetrated through the alimentary tract in the neck region and entered the hemocoel. A melanization reaction killed many invading nematodes, but heavier concentrations overwhelmed the hosts' defense reaction and 100% mortality of third- and fourth-instar larvae was achieved using between 170 and 200 nematodes per host. Death was either due to the nematode releasing cells of the symbiotic bacterium, Xenorhabdus luminescens, into the hemocoel or to foreign bacteria (mostly Pseudomonas aeruginosa), which were introduced by the penetrating nematodes. The potential use of this nematode as a biological control agent of larval culicine mosquito is discussed.     

Tipula iridescent virus infection in the developmental stages of Tipula oleracea

Tipula iridescent virus (TIV) is infective to all four larval instars, pupae, and adults of both sexes of Tipula oleracea, and iridescence has been observed in infected insects at all these stages. Third- and fourth-instar larvae were more resistant to ingested TIV than first and second instars. When TIV was injected into the hemocoel, the results suggested a possible decrease in resistance from the third larval instar to the pupa. Incubation periods (times from injection of TIV to appearance of iridescence) were significantly shorter in older fourth-instar larvae than in younger fourth-instar or thirdinstar larvae, but variability in incubation period was significantly greater in younger fourth-instar larvae than in the other two stages. Many insects which were inoculated with TIV in one stage developed iridescence and died in later stages. The amounts of infective TIV in two infected adults were estimated.     

Brain-independent development in the moth Sesamia nonagrioides

The caterpillars of Sesamia nonagrioides developing under long-day (LD) photoperiod pupate in the 5th or 6th instar whereas under short day (SD) conditions they enter diapause and undergo several extra larval molts. The diapause is terminated within 1-3 instars upon transfer of SD larvae to the LD conditions. Brain removal from the 6th instar larvae promotes pupation followed by imaginal development; however, one third of the SD larvae and 12% of the LD larvae debrained at the start of the instar first undergo 1-2 larval molts. The incidence of larval molts is enhanced by the brain implants. Exclusively pupal molts occur in the LD larvae debrained late in the 6th instar. Decapitation elicits pupation in both LD and SD larvae, except for some of the 4th and 5th and rarely 6th instar that are induced to a fast larval molt. The pupation of decapitated larvae is reverted to a larval molt by application of a juvenile hormone (JH) agonist. No molts occur in abdomens isolated from the head and thorax prior to the wandering stage. Abdomens isolated later undergo a larval (SD insects) or a pupal (LD insects) molt. Taken together the data reveal that in S. nonagrioides (1) several larval molts followed by a pupal and imaginal molt can occur without brain; (2) an unknown head factor outside the brain is needed for the pupal-adult molt; (3) brain exerts both stimulatory and inhibitory effect on the corpora allata (CA); (4) larval molts induced in CA absence suggest considerable JH persistence.     

Biosteres longicaudatus: Developmental dependence on host (Anastrepha suspensa) physiology

Larvae of Anastrepha suspensa that were in the first day of the third instar were parasitized by females of the solitary endoparasitoid, Biosteres longicaudatus. At the end of the 6-hr oviposition period, larvae were ligated posterior to the ring gland so that some larvae had parasitoids anterior to the ligature while in others, the parasitoids were in the abdomen, posterior to the ligature. Ninety-two percent of the parasitoids anterior to the ligature hatched to the first through third instars. Parasitoids posterior to the ligature had a 75% egg hatch to the first instar only. No larval molts to the second or subsequent instars occurred in these parasitoids. Upon parabiosis to 3-day-old, unparasitized host pupae, the ligated larvae pupated and 97% of the first-instar parasitoids in these parabiosed larval abdomens molted to the second instar. Newly laid parasitoid eggs transplanted to 3-day-old pupal hosts had less than one-third of the egg hatch of those transplanted to first-day third-instar hosts. The data implicate the physiological state of the host (vis-a-vis pupation and associated events) as being an important factor in the development of the endoparasitoid.     

Effect of starvation upon baculovirus replication in larval Bombyxmori and Heliothisvirescens

The progression of baculovirus (BmNPV, BmCysPD, AcMNPV or AcAaIT) infection in larval Bombyx mori and Heliothis virescens (1st, 3rd or 5th instar) was investigated following various starvation regimes. When the larvae were starved for 12 or 24 h immediately following inoculation, the median lethal time to death (LT₅₀) was delayed by 9.5-19.2 h in comparison to non-starved controls. This corresponded to a delay of 10-23% depending upon the larval stage and virus that was used for inoculation. When a 24 h-long starvation period was initiated at 1 or 2 days post inoculation (p.i.), a statistically significant difference in LT₅₀ was not found indicating that the early stages of infection are more sensitive to the effects of starvation. Viral titers in the hemolymph of 5th instar B. mori that were starved for 24 h immediately following inoculation were 10-fold lower (p < 0.01) than that found in non-starved control larvae. Histochemical analyses indicated that virus transmission was reduced in 5th instar B. mori that were starved for 24 h immediately following inoculation in comparison to non-starved control larvae. In general, the mass of larvae that were starved immediately after inoculation was 30% lower than that of non-starved control insects. Our findings indicate that starvation of the larval host at the time of baculovirus exposure has a negative effect on the rate baculovirus transmission and pathogenesis.     

The comparative susceptibilities of Pieris brassicae and P. rapae to a granulosis virus from P. brassicae

A comparison was made of the dosage-mortality responses of larvae of Pieris brassicae and P. rapae to infection by P. brassicae granulosis virus (GV). Bioassays with first, second, third, and fourth-instar larvae of both species revealed a marked difference in susceptibility between instars and between species. Median lethal dosages (LD₅₀s) for P. rapae larvae ranged from five capsules for the first instar to 662 capsules for the fourth instar. With P. brassicae, this range extended from 66 capsules to 2.3 × 10⁷ capsules. The time-mortality responses of the two species were similar when fed virus dosages equivalent to an LD₉₀. Median lethal times (LT₅₀s) ranged from 5 days for first-instar larvae to 7–8 days for fourth-instar larvae. A comparison between a long-established laboratory stock of P. brassicae and a stock recently acquired from the field showed no significant difference in their susceptibility to GV. The implications of the pronounced species differences in susceptibility to GV infection are discussed in relation to the potential field control of P. rapae and P. brassicae.     

A recombinant immunosuppressive protein from Pimpla hypochondriaca (rVPr1) increases the susceptibility of Lacanobia oleracea and Mamestra brassicae larvae to Bacillus thuringiensis

The precise mechanisms underlying Bacillus thuringiensis-mediated killing of pest insects are not clear. In some cases, death may be due to septicaemia caused by Bt and/or gut bacteria gaining access to the insect haemocoel. Since insects protect themselves from microbes using an array of cellular and humoral immune defences, we aimed to determine if a recombinant immunosuppressive wasp venom protein (rVPr1) could increase the susceptibility of two pest Lepidoptera (Lacanobia oleracea and Mamestra brassicae) to Bt. Bio-assays indicated that injection of 6 μl of rVPr1 into the haemocoel of both larvae caused similar levels of mortality (less than 38%). On the other hand, the LD_30-40 of Bt for M. brassicae larvae was approximately 20 times higher than that for L. oleracea larvae. Furthermore, in bio-assays where larvae were injected with rVPr1, then fed Bt, a significant reduction in survival of larvae for both species occurred compared to each treatment on its own (P < 0.001); and for L. oleracea larvae, this effect was more than additive. The results are discussed within the context of insect immunity and protection against Bt.     

Soybean leaf consumption by Nomuraea rileyi (Fungi: Deuteromycotina)-infected Plathypena scabra (Lepidoptera: Noctuidae) larvae

To determine the impact of Nomuraea rileyi on consumption by the green cloverworm, Plathypena scabra, larvae were reared from eggs obtained from field-collected moths, inoculated with conidia, and placed individually in separate plastic vials with a piece of surface-sterilized soybean leaflet. No significant differences in consumption rates were found between N. rileyi-inoculated and control larvae until after 144 hr post-treatment. After this period, consumption by larvae inoculated as first, second, third, or fourth instars was significantly less than that of control larvae because of mortality. First or second instars inoculated with N. rileyi conidia had significantly longer subsequent stadia than did control larvae. The average LT₅₀ of all inoculated instar groups was 6.5 days, and there was little evidence of significantly different N. rileyi susceptibility among instar groups. In general during the N. rileyi infection period leading to their deaths, inoculated P. scabra larvae had consumption patterns that were very similar to those of healthy larvae.     

Dry-matter budgets for Diacrisia casignetum larvae fed on sunflower leaves

Larvae of Diacrisia casignetum Kollar were reared in groups of 100, 50 and 10 on fresh sunflower leaves at 27 ± 1°C temperature, 75 ± 5% r.h. and 12 h light each day. Rates of feeding, excretion and weight gain increased with age of the larva and were greatest in the sixth instar. Differences in these rates attributable to density were statistically heterogeneous (P < 0.001) whereas, of those attributable to instar, the weight gain was significant (P < 0.10). The second-instar larvae had highest levels of protein and carbohydrates in the body tissues; and the sixth-instar larvae had, the least. The pattern of protein assimilation in various instars was similar to that of glycogen except that proportional differences in the latter were pronounced in fifth and sixth instars. Consumption index, growth rate and approximate digestibility were high in the early instars whereas efficiencies of conversion of ingested and digested food were high in advanced instars. Food utilization, as reflected by efficiency of conversion of ingested and digested values in advanced instars, indicated the amount needed for maintenance decreased and more energy was left for growth and reproduction.     

A laboratory evaluation of the pathogenicity of Bacillus popilliae var. rhopaea, the agent of milky disease in Rhopaea verreauxi (Coleoptera: Scarabaeidae)

Two methods of infection, i.e., feeding known numbers of spores and rearing larvae in contaminated peat, were used to bioassay the susceptibility of Rhopaea verreauxi to Bacillus popilliae var. rhopaea at 23°C. The susceptibility of the three larval instars was similar as measured by the ID₅₀ and IC₅₀ values. However, within an instar, newly molted larvae were less susceptible than mature larvae when infected by the contaminated peat method. It is suggested that this was due to reduced food intake. The range of ID₅₀ values for all bioassays with R. verreauxi larvae were 1.1 × 10⁷ to 4.0 × 10⁷ spores per larva, and IC₅₀ values were 3.4 × 10⁶ to 5.0 × 10⁷ spores per g of contaminated peat. The slope of the probit line was always low (0.6 to 1.8) except for young first-instar larvae infected by contaminated peat when the slope was 4.0. Disease per se did not affect food intake, though intake was reduced at high doses of contaminated peat. Young larvae often died without developing symptoms but, with increasing age, infected larvae were more likely to develop symptoms. Bioassays with Othnonius batesi and Rhopaea morbillosa indicated a much lower susceptibility per os than for R. verreauxi. It is concluded that the potential for using B. popilliae var. rhopaea to control R. verreauxi is high, but the bacillus is unlikely to be of value in control of O. batesi or R. morbillosa.     

Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its host L. dispar. L. dispar exhibits midgut-based and systemic, age-dependent resistance to LdMNPV within the fourth instar; the LD₅₀ in newly molted larvae is approximately 18-fold lower than in mid-instar larvae (48-72 h post-molt). We examined the role of the immune system in systemic resistance by measuring differences in hemocyte immunoresponsiveness to foreign targets, hemolymph phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD) activities, and melanization of infected tissue culture cells. Mid-instar larvae showed a higher degree of hemocyte immunoresponsiveness, greater potential PO activity (pro-PO) at the time the virus is escaping the midgut to enter the hemocoel (72 h post-inoculation), greater GLD activity, and more targeted melanization of infected tissue, which correlate with reduced viral success in the host. These findings support the hypothesis that innate immune responses can play an important role in anti-viral defenses against baculoviruses and that the success of these defenses can be age-dependent.     

Effect of Host Plant on the Infectivity of Sl MNPV to Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) Larvae

The susceptibility of Spodoptera litura to SlMNPV infection was markedly affected by phyto-chemicals ingested during the acquisition of viral inoculum on foliage of tomato and cauliflower. The LD₅₀ values computed for second, third and fourth instar larvae assayed on tomato leaves were 254, 819 and 23395 PIBs/larva, respectively whereas, it was 326, 1719 and 43843 PIBs/larva for respective instars when assayed on cauliflower leaves. Thus LD₅₀ values for second, third and fourth instar larvae were 1.28-, 2.09- and 1.87- fold lower, respectively in tomato leaves. Similarly, LT₅₀ values for second, third and fourth instar larvae assayed on tomato leaves were 7.1 and 7.5 days, respectively at inoculum dose of 2.7×10⁴ PIBs/larva whereas, it was 7.7 and 8.0 days for respective instars when assayed on cauliflower leaves at same inoculum. This result also showed that the S. litura were more susceptible on tomato leaves in comparison to cauliflower leaves as the time required for mortality was lower in tomato leaves. The possible biochemical bases for differential level of mortality of S. litura larvae on tomato and cauliflower crops needs to be investigated.     

Chemical allatectomy of late Locusta embryos by a synthetic precocene and its effect on hopper morphogenesis

The anti-allatin substance, 7-ethoxy-precocene II (= “precocene III”) was topically applied to eggs of Locusta migratoria migratorioides with fully grown embryos in stage XX (about 64 ± 4% of the whole period of egg development). A day after precocene application the eggs were washed for 10–15 s in acetone and then transferred to clean containers for removing precocene residues and for preventing contamination at hatching. The treatment induced prothetelic morphogenetic disturbances which became overt in the subsequent hoppers; the effect was dose dependent and the ED₅₀ (= effective anti-allatin dose 50%) was low, 20.5 μg precocene III per g fresh egg weight (= 0.37 μg per egg). Quite similar results were obtained following application of precocene III to eggs with embryos in stage XXI (73 ± 4% of the egg development). These findings and direct examination of histological sections of the embryonic corpora allata demonstrated that precocene chemically allatectomizes late Locusta embryos. The lethal effect of precocene III was dependent on the washing. When the eggs were washed in acetone a day after application, mortality did not occur in a dose-dependent way; even the highest dose applied, 256 μg precocene III per egg (= 14405 μg per g fresh weight), was less than the LD₅₀ (lethal dose 50%). In contrast, without washing mortality was dose dependent, but it occurred later, at or after hatching; the LD₅₀ was 1334.9 μg per g (= 22.7 μg/egg). The results show that the late embryos are highly susceptible to the anti-allatin effect of the precocene, but are extremely insusceptible to its lethal effect; toward hatching, however, susceptibility to the lethal effect becomes marked.With doses between 45–14405 μg precocene III per g fresh egg weight, the anti-allatin effect became overt by a quite-uniform belated morphogenetic response. All hoppers which hatched from precocenetreated eggs were morphogenetically normal in the 1st instar and in the beginning of the 2nd instar, but the duration of the 2nd instar was almost doubled and at the end of this instar over 96% of the locusts died in the moult, being unable to shed the exuvia. Artificial removal of the apolyzed old cuticle revealed 3rd instar prothetelic adultiforms. These results and some data in the literature indicate that allatectomy of the embryo does not result in prothetelic morphogenetic disturbances in the 1st and early 2nd instar larvae and may impose the question what is the role of the juvenile hormone in late embryos and early larvae.     

Susceptibility of the spruce budworm, Choristoneura fumiferana, and the white-marked tussock moth, Orgyia leucostigmata, to Bacillus thuringiensis: Chemical insecticide combinations

Bacillus thuringiensis mixed with the organophosphate insecticides, fenitrothion (Sumithion), Gardona^®, and Orthene^®, or the synthetic pyrethroid, SBP 1382, was incorporated into synthetic diet and fed larvae of the spruce budworm, Choristoneura fumiferana, and the white-marked tussock moth, Orgyia leucostigma. Mortality was highest when larvae were fed combinations of low concentrations of the insecticides and low to moderate concentrations of the pathogen. The data indicated that applications of a B. thuringiensis dosage expected to produce about 45% mortality of third and fourth instar larvae of the spruce budworm combined with a dosage of fenitrothion causing about 40% mortality or a dosage of Orthene causing from 5 to 25% mortality should result in low budworm survival. With a B. thuringiensis dosage causing 20–60% mortality combined with a fenitrothion dosage causing 15–50% mortality or a sublethal dosage of Gardona, a low survival rate of young white-marked tussock moth larvae may be expected.     

The dependence of development and fecundity of Samea multiplicalis on early larval nitrogen intake

Larvae of the moth Samea multiplicalis (Lepidoptera: Pyralidae), developed more rapidly when their food plant, Salvinia molesta was richer in nitrogen. Larvae that fed on plants with less than 1.35% nitrogen during their first instar completed the larval stage in 18.0–24.2 days, after passing through six instars. In contrast, larvae that had fed on food richer in nitrogen during their first instar completed the larval stage in 14.1–17.4 days, and passed through five instars. Experience of nitrogen in later instars had little additional effect on total larval development time. Oöcyte complements of 1-day old adult females was correlated with the mean food nitrogen content over the larval period. Food nitrogen content experienced by larvae in earlier instars was as important as that in the final instars, in determining number of mature oöcytes, which suggests early conditioning of the nitrogen use effciencies of later instars.     

Morphological diversity of first instar larvae in Miltogramma subgenus Pediasiomyia (Diptera: Sarcophagidae, Miltogramminae)

The morphology of first instar larvae of three species of Miltogramma Meigen subgenus Pediasiomyia Rohdendorf is described using SEM and light microscope techniques. Miltogramma chrysochlamys (Rohdendorf), Miltogramma margiana (Rohdendorf) and Miltogramma przhevalskyi (Rohdendorf) share with other satellite flies the presence of an elongated sensillum basiconicum of the maxillary palpus and numerous longitudinal cuticular ridges on the integument of all segments; and they share with other Miltogramma spp. a maxillary palpus situated on a more or less raised base. While the first instar M. margiana is very similar to first instars of Miltogramma (sensu stricto), first instars of M. chrysochlamys and M. przhevalskyi share several features unique among Miltogramminae: antennal dome situated on a flat or indistinct basal ring, antennal dome flattened, border between pseudocephalon and first thoracic segment with a pair of fleshy processes, and basal ring with trichoid sensillum. First instars of M. chrysochlamys and M. przhevalskyi are unique in having both sb1 and sb2 of the sensilla basiconica of the maxillary palpus elongated, and M. przhevalskyi has an unpaired, median proleg on most abdominal segments.     

The effect of juvenile hormone on prothoracic gland function during the larval-pupal development of Manduca sexta: An in situ and in vitro analysis

The application of juvenile hormone I or ZR 512 to neck-ligated, day-5 fifth instar (V₅) larvae reduced the time to pupation in a dose-dependent manner when compared to neck-ligated controls treated with methyl epoxy stearate. Haemolymph ecdysteroid titres determined by radioimmunoassay (RIA) reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal development, i.e. the ecdysteroid titres were similar to those of normally developing larvae although the ecdysteroid peak elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged the ecdysteroid peak in normal larvae by 2 days. Neck-ligated V₅ larvae that were untreated ultimately pupated and the haemolymph ecdysteroid peak eliciting pupation in these animals was 7 μg/ml haemolymph, almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated larvae. The data indicated that juvenile hormone I does stimulate the prothoracic glands but to determine whether this stimulation was direct or indirect, an in vitro approach was taken. Prothoracic glands from V₅, V₆ and V₇ larvae were incubated in vitro under conditions in which they could be stimulated by prothoracicotropic hormone, and were exposed to concentration of free juvenile hormones I, II, III or ZR 512 ranging from 10^?5M to 10^?10M. In no case were the prothoracic glands stimulated in a dose-dependent manner that would be indicative of hormone activation. Similar results were obtained when juvenile hormone bound to binding protein was incubated with the prothoracic glands. Studies with the acids of the three juvenile hormone homologues revealed them to be ineffective in activating prothoracic glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone by day-0 pupal prothoracic glands. The significance of the latter effect is unknown. It is concluded from these data that juvenile hormone can, indeed, activate late larval prothoracic glands in situ, but does so indirectly.     

Cold-induced glycerol accumulation by Ostrinia nubilalis larvae is developmentally regulated

Ostrinia nubilalis larvae reared under both nondiapause and diapause-inducing conditions were chilled at 5°C for various periods and their haemolymph glycerol concentrations were measured enzymatically. The ability of fifth (final) instars to accumulate glycerol was dependent upon cold stress but not the diapause state. Furthermore this response was independent of any cold-induced release of cephalic or thoracic hormones. The capacity of O. nubilalis larvae to express cold-induced glycerol accumulation was found to require ecdysis from the fourth to fifth instar. Eggs as well as second, third and fourth instars were completely incompetent. These results indicate that, at the biochemical level, a specific developmental programme or sequence is required for O. nubilalis to demonstrate this response to cold stress.     

Bursicon-mediated control of tanning in melanizing and non-melanizing first instar larvae of Schistocerca gregaria

Both melanization and sclerotization in first instar Schistocerca larvae are shown to have a common hormonal control mechanism. This hormone is shown to be analogous to bursicon, the tanning hormone of other insects, by the standard blowfly bioassay. The hormone is identical in both melanizing and non-melanizing first instar larvae of Schistocerca.     

Effects of the parasitization of a braconid, Apanteles, on the blood of its host, Pieris

Parasitization of a braconid wasp, Apanteles glomeratus, of larvae of a common cabbage butterfly, Pieris rapae crucivora, caused changes in differential haemocyte count (DHC), total haemocyte count (THC), and encapsulative capacity against dead eggs of Apanteles in the fourth and fifth instar host larvae.However, no correlation could be found between the number of Apanteles eggs deposited and THC of the middle fourth instar host larvae or between the number of parasitoid larvae and specific gravity of the haemolymph from the late fifth instar host larvae.From the changes in DHC and in THC of both non-parasitized and parasitized Pieris larvae, an increase in the number of plasmatocytes of non-parasitized Pieris larvae in the early fourth instar period was supposed to be due to transformation of prohaemocytes into plasmatocytes, and a low population of plasmatocytes of parasitized larvae in the comparable period was assumed to be due to a suppression of transformation of prohaemocytes by some factor released from the parasitoid eggs.Failure of the parasitized fourth instar Pieris larvae to encapsulate injected dead eggs of Apanteles indicated that the parasitoid embryos were, in some way, actively inhibiting the encapsulation reactions of the host.The increase in THC of the parasitized fifth instar larvae could not be ascribed to a decrease in the volume of host haemolymph. Rather it could be interpreted by a suppression of adhesive capacity of haemocytes in the host haemocoel to tissue surfaces.Reduced encapsulative capacity of the parasitized fifth instar larvae might be attributed either to a depression of the adhesive activity of plasmatocytes resulting from a depletion of energy source for haemocytes in the host haemolymph by parasitization, or from an active suppression of adhesiveness of the plasmatocytes by secretions from ‘giant cells’ (teratocytes) originated from the parasitoid.