Responses of the entomopathogenic nematode, Steinernema riobrave to its insect hosts, Galleria mellonella and Tenebrio molitor.

Potential hosts for infective juveniles of entomopathogenic nematodes can vary considerably in quality based on the characteristics of the host species/stage, physiological status (e.g. stress, feeding on toxins), and infection status (heterospecific or conspecific infection). In this study, we investigated responses of the entomopathogenic nematode Steinernema riobrave to hosts (Galleria mellonella or Tenebrio molitor) that were previously parasitized with conspecifics or injected with the nematode-symbiotic bacterium, Xenorhabdus sp., to determine if there is a preference for previously parasitized/injected hosts and when this preference might occur. In no-choice bioassays, the number of juveniles infecting both host species decreased with increasing time post-infection. However, infective juveniles continued to infect previously parasitized hosts up to 72 h. Significant preference was exhibited by S. riobrave for 24 h post-infection G. mellonella larvae over uninfected, and by 24 h post-injection G. mellonella larvae over 48 h post-injection larvae. No significant preference was exhibited by S. riobrave for T. molitor hosts previously parasitized with conspecifics or those injected with bacteria in any treatment combination. Such preference for, or continued infection of parasitized insects, has the potential to impact nematode efficacy.

Attraction behaviour of three entomopathogenic nematode species towards infected and uninfected hosts.

Entomopathogenic nematode infective juveniles are likely to encounter both uninfected and infected insects and host quality depends on the stage of the infection. We hypothesized that nematode response to infected hosts will change over the course of an infection. Here, we tested this hypothesis by focusing on the influence of host infection status on long-range attraction to host volatile cues. The attraction response of 3 nematode species (Steinernema carpocapsae, S. glaseri and S. riobrave) with different foraging strategies to infected and uninfected insects (Galleria mellonella and Tenebrio molitor) was tested at 24 h intervals from start of infection to emergence of infective juveniles from depleted host. As expected, based on their foraging strategies, S. carpocapsae was not very responsive to hosts, S. glaseri was highly responsive and S. riobrave was intermediate. Generally, the level of attraction did not change with time after infection and was similar between infected and uninfected hosts. An exception was S. glaseri infected T. molitor, which tended to be less attractive to S. glaseri than uninfected hosts. These results suggest that any influence of host infection status on infection behaviour is occurring at subsequent steps in the host-infection process than host attraction, or involves non-volatile cues.

Parasitoid-host endocrine relations: self-reliance or co-optation?

High titers of juvenile hormone (JH) maintain developmental arrest in Manduca sexta larvae parasitized by Cotesia congregata. Parasitized hosts exhibit up to 9.5 times greater amounts of total hemolymph JH (from 0.6+/-0.09 to 2.51+/-0.43ng/ml) compared to non-parasitized controls. Elevated titers are observed throughout the fifth instar, even beyond egression of the parasitoids on day 5. GC-MS analysis revealed that in hemolymph of unparasitized control larvae, JH I is the major homolog and levels of JH III are negligible; in parasitized individuals the amounts of JH I, II, and III rise, and JH III predominates. Neck ligation ensured separation of M. sexta's corpora allata from the posterior section, which contained most of the parasitoids in the infected insects. When the posterior region was sampled, JHs were not detected in the non-parasitzed larvae, but in those parasitized, JH III was found (1.98+/-0.29ng/ml, 24 h post-ligation). JH III was the only homolog produced and secreted by the parasitoid in in vitro culture. This is the first report stating that a parasitoid secretes JH III and may contribute, at least in part, to the circulating titer in the host hemocoel, concurrently promoting host production of JH I and II.

Sex pheromones of Callosobruchus subinnotatus and C. maculatus (Coleoptera: Bruchidae): congeneric responses and role of air movement.

Females of Callosobruchus spp. are known to produce sex pheromones that attract males. These sex pheromones cannot be adopted for use in pest management without first investigating the responses of the males in the windless conditions of storage environments. Consequently, behavioural bioassays of Callosobruchus subinnotatus Pic males were conducted in an olfactometer in the absence of air-flow. Under these conditions males were found to be able to follow odour trails to the source. However, the latency period was longer in diffusional bioassays than for insects in a Y-tube olfactometer that provided directional wind cues. The highest percentage of males reached the pheromone source when components of the pheromones, (E)-3-methyl-2-heptenoic acid (E32A) and (Z)-3-methyl-2-heptenoic acid (Z32A), were formulated in a 50:50 or 25:75 ratio. Males of C. maculatus (Fabricius) responded to sex pheromone of C. subinnotatus, but males of C. subinnotatus did not respond to that of C. maculatus. The two sex pheromone components of C. subinnotatus are also constituents of C. maculatus sex pheromone. These two components may be potentially useful in monitoring the populations of both species in stored beans. It is postulated that (Z)-3-methyl-3-heptenoic acid (Z33A), the major component of the sex pheromone of C. maculatus, must have acted as an antagonist inhibiting response of C. subinnotatus to the sex pheromone of C. maculatus.

Role of juvenile hormone-esterase in mating-stimulated egg development in the moth Heliothis virescens.

Juvenile hormone (JH) titer in virgin females of Heliothis virescens is significantly lower than that in mated females of the same age. The JH titer in virgin females follows a diel pattern in which it begins to increase towards the end of photophase, remains high around the onset of scotophase, and declines during scotophase. The titer reaches its lowest levels at the onset of photophase, and remains low during the first half of photophase. In mated females, the diel pattern of JH titers is not as pronounced. JH-esterase (JHE) activity in mated females is significantly lower than that of virgin females during photophase; JHE levels in the former are similar to levels seen in newly emerged females. JHE activity in mated females also exhibits a diel pattern, in which activity is low during photophase and high at the onset of scotophase. Evidence for the indirect involvement of JHE in the mating-stimulated egg development is provided by the effect of selected JHE inhibitors in inhibiting JHE activity and stimulating egg production in virgin females.

Choristoneura fumiferana entomopoxvirus prevents metamorphosis and modulates juvenile hormone and ecdysteroid titers.

Larvae of the spruce budworm, Choristoneura fumiferana, infected with C. fumiferana entomopoxvirus (CfEPV) continue to feed and grow without undergoing metamorphosis and die as moribund larvae. The lethal dose (LD(50)) and lethal time (LT(50)) values for fourth instar larvae are 2.4 spheroids and 25.2 days, respectively. One hundred percent of the control fourth instar larvae, which were fed water instead of virus, pupated by 18 days post feeding (PF). Only 30% of the larvae that were fed the LD(50) dose and none of the larvae that were fed the LD(95) dose pupated by 18 days PF. Of the control larvae, 95% became adults by 24 days PF, whereas in the treated group only 2% of larvae that were fed the LD(50) dose and none of the larvae that were fed the LD(95) dose became adults by 24 days PF. Some of the virus-treated larvae died as either larval/pupal or pupal/adult intermediates. These phenotypic effects were similar to the larval/pupal and pupal/adult intermediates, resulting from treating larvae with juvenile hormone (JH) or its analogs, which suggests that EPV may cause such abnormalities by modulating JH and/or ecdysteroid titers. In untreated sixth instar larvae the JH titer decreased to low levels by 24 h after ecdysis and remained low throughout larval life. EPV-fed sixth instar larvae had 2112 pg/ml on day 0, 477 pg/ml on day 1 and 875 pg/ml on day 8 of the sixth instar. Control larvae contained 860 ng of ecdysteroids per ml hemolymph on day 8 of the sixth instar, whereas EPV-treated larvae of the same age (30 days PF) had only 107 ng of ecdysteroids per ml of hemolymph. Thus, EPV infection results in increased JH titer and decreased ecdysteroid titer. Northern hybridization analysis was performed using RNA isolated from control and EPV-fed larvae and cDNA probes for (i) juvenile hormone esterase (JHE), which is JH inducible, (ii) Choristoneura hormone receptor 3 (CHR3), which is ecdysteroid inducible, and (iii) larval specific diapause associated protein 1 (DAP1), whose expression is larval specific. EPV-treated larvae showed higher levels of JHE and DAP1 mRNA and lower levels of CHR3 mRNA, indicating that they had higher levels of JH and lower levels of ecdysteroids. Thus, our data show that EPV prevents metamorphosis by modulating ecdysteroid and JH levels.

Lipophorin of female Blattella germanica (L.): characterization and relation to hemolymph titers of juvenile hormone and hydrocarbons.

High density lipophorin (HDLp) from the hemolymph of the German cockroach, Blattella germanica (L.) (Family Blattellidae), has an apparent molecular weight of 670kDa, with an isoelectric point of 7.0 and a density of 1.109g/ml. It is composed of two subunits, apolipoprotein-I (212kDa) and apolipoprotein-II (80kDa), and consists of 51.4% lipid, 46.2% protein and 2.4% carbohydrate. Hydrocarbons constitute 42.2% of the total lipids which also contain diacylglycerol, cholesterol and phospholipid. Lipophorin is rich in the amino acids glutamic acid, aspartic acid, lysine, valine, and leucine. Specificity of a polyclonal antibody was demonstrated by Western blotting and Ouchterlony immunodiffusion: the antiserum recognized native HDLp and apolipoprotein-I, but not apolipoprotein-II, purified vitellin, or other hemolymph proteins. It also recognized a protein in the hemolymph of Supella longipalpa (Blattellidae) but did not cross-react with hemolymph proteins from Periplaneta americana (Blattidae) or Diploptera punctata (Blaberidae). An enzyme-linked immunosorbent assay was developed to measure the HDLp titer in the hemolymph of adult females. The titer of HDLp, a juvenile hormone binding protein, exhibited no clear relationship to the changing titer of juvenile hormone in hemolymph. The hemolymph titer of hydrocarbon, which is also carried by HDLp, showed some functional relation to the concentration of HDLp in the hemolymph. Because it concurrently serves multiple functions in insect development and reproduction, lipophorin titer might covary with the titers of lipid ligands that occur at high concentrations and require extensive shuttling through the hemolymph.

Mating in Heliothis virescens: transfer of juvenile hormone during copulation by male to female and stimulation of biosynthesis of endogenous juvenile hormone.

Studies were undertaken to determine whether adult males of Heliothis virescens transfer juvenile hormone (JH) to females during copulation, and an in vitro radiochemical assay was used to determine whether mating causes an allatotropic effect, i.e., stimulation of JH biosynthesis by corpora allata (CA). In vitro, CA from 3-day-old mated females synthesized and released approximately 2.5 times total JH as that of CA from comparably aged virgin females. Of the homologues, JH II exhibited significant increase in mated females; JH I also increased but not significantly. JH III remained similar to that of virgin females. This is the first demonstration of an allatotropic effect of mating in moths. In contrast to the female, CA of virgin males did not produce any JH, but accessory sex glands (ASG) in 3-day-old males synthesized small amounts of JH. Immediately after adult emergence, male ASG contained approximately 1.5 ng JH I and II, which increased by 12 h after emergence and remained at this high level up to 54 h after emergence. JH III was barely detected in ASG. JH in ASG of mated male immediately after uncoupling was depleted almost completely, and 24 h later recovered to levels comparable to that of 54-h-old virgin male. Virgin female bursa copulatrix did not contain any JH, but mated female bursa, immediately after uncoupling, had JH at levels comparable to that observed in virgin male ASG. By 6 h after uncoupling, JH levels decreased dramatically in mated female bursa. These data suggest the transfer of JH to females by the male.

Temporal profiles of juvenile hormone titers and egg production in virgin and mated females of Heliothis virescens (Noctuidae).

Juvenile hormones (JH) I, II, and III were monitored in hemolymph of virgin and mated females of various ages in Heliothis virescens. JH I was the predominant homologue followed by JH II, but JH II was present at a higher level in young virgin females. JH III was detectable only at a low level. In virgin females, hemolymph JH titers were low at emergence (2.2ng/ml-total amount of JH I and JH II), but increased thereafter and reached a maximum at 24h of age (53.5ng/ml). At 30h and 36h of age, JH titers dropped to a low level, but increased again in older virgin females. After mating, JH titers increased significantly. JH titers at 0h after uncoupling (137.4ng/ml) were nearly 3 times as high as those in 24-h-old virgin females. Within 6h after uncoupling, JH titers decreased slightly, but titers increased with age of mated females and reached a level of 320.2ng/ml hemolymph at 72h after uncoupling. The titer of JH I and JH II was correlated highly with total number of eggs produced (r(2)=0.70, P<0.001). Mating stimulated JH production, resulting in an increase in egg production.

Spermatophore formation and regulation of egg maturation and oviposition in female Heliothis virescens by the male.

This study was undertaken, using various surgical manipulations, to examine the role and functions of accessory sex glands and testes in Heliothis virescens in spermatophore formation and stimulation of egg maturation in mated females. Normal females mated to accessory sex glandectomized (-ASG) males produced fewer eggs and retained most of their eggs in ovarioles compared with females mated to sham-operated (+ASG) or normal males. The ASG are the source of the components for spermatophore formation in H. virescens. Females mated to castrated (-Testes) males showed similar pattern of egg production as did females mated to -ASG males. Females mated to -Testes males and those mated to sham-operated (+Testes) or normal males were found to have spermatophore in the bursa after uncoupling. Normal females mated to allatectomized (-CA) males developed similar numbers of total eggs as did females mated to sham-operated (+CA) males and normal males.

Hemolymph juvenile hormone titers in pupal and adult stages of southwestern corn borer [Diatraea grandiosella (pyralidae)] and relationship with egg development.

Juvenile hormones I, II and III were monitored in hemolymph of pupal and adult stages of various ages of Diatraea grandiosella females. JH III was the predominant homologue followed by JH II, and JH I was rarely detectable. At day 5 after pupation, no JH was detectable. JH titers increased from 7.5days after pupation to a peak of 24.8ngml(-1) JH II and 26ngml(-1) JH III at adult emergence and then declined to low levels by 24h after emergence. Ovarian development in D. grandiosella parallels changes in hemolymph JH titers, but the role of JH in vitellogenesis is unclear since the time of vitellogenesis initiation has yet to be determined. No apparent vitellogenin deposition was observed in eggs 5days after pupation. Some oocytes were partially vitellogenic by 7.5days after pupation and oocytes continued to grow afterwards, but no oocytes were chorionated during the pupal stage. Chorionated oocytes were observed in 24-h-old female moths. Juvenile hormone is essential for chorion formation in this species, because decapitated pupae treated with 10&mgr;g JH III in corn oil developed chorionated oocytes while decapitated pupae treated with corn oil did not.

Pheromonotropic and pheromonostatic activity in moths.

Pheromone biosynthesis in many species of moths requires a pheromonotropic neurosecretion, the pheromone biosynthesis activating neuropeptide (PBAN), from the brain-subesophageal ganglion-corpora cardiaca complex. Some investigations suggest that PBAN is released into the hemolymph and acts directly on sex pheromone glands (SPG) via a Ca++/calmodulin-dependent adenylate cyclase. Others suggest, however, that PBAN acts via octopamine that is released by nerves from the terminal abdominal ganglion innervating the SPG. These findings suggest that there are controversies on the mode of action of PBAN and other pheromonotropic factors, sometimes even within the same species. Mating in many insects results in temporary or permanent suppression of pheromone production and/or receptivity. Such a suppression may result from physical blockage of the gonopore or deposition of pheromonostatic factor(s) by the male during copulation that result in suppressed pheromone production and/or receptivity in females either directly or by a primer effect. In several species of insects, including moths, a pheromonostatic factor is transferred in the seminal fluid of males. Similar to the controversies associated with the pheromonotropic activity of PBAN, sometimes even within the same species, there appear to be controversies in pheromonostasis in heliothines as well. This paper reviews these conflicting findings and presents some data on pheromonostatic and pheromonotropic activity in Heliothis virescens that support and conflict with current information, raising further questions. Answers to some of the questions are partly available; however, they remain to be answered unequivocally.

Rate of release of spruce budworm pheromone from virgin females and synthetic lures.

The rate of pheromone [(E)- and (Z)-11-tetradecenal] release from calling virginChoristoneura fumiferana females and synthetic lures was determined in both static and aerated atmospheres. In a static system ca. 2 ng/hr was recovered per female. Owing to the > 75% adsorption onto the females' bodies in static atmospheres, the actual release rate has to be corrected to roughly 9-27 ng/hr, depending on the percentage adsorbed. In the air-flow system, females were found to release between 4 and 20 ng/hr. On a 16∶8 light-dark cycle, calling began 1-2 hr before lights-off and continued nonstop until lights-on. Pheromone was emitted throughout calling, while no pheromone was detected during the noncalling periods.

Sensilla of the antennae and the labial and maxillary palps of Blattella germanica (L.) (Dictyoptera: Blattellidae): Their classification and distribution.

The various sensilla on the antennae and on the labial and maxillary palps of Blattella germanica (L.) were studied. Thick-walled chemoreceptors with fluted shafts and articulated bases are located on the antennae and on the labial and maxillary palps. Thin-walled chemoreceptors, without fluted shafts or articulated bases, are restricted to the flagellar segments of the antennae and to the distal segments of the palps. Antennae of adult males have more thin-walled chemoreceptors than do those of females. Hair-plate sensilla are found at the scape-head and scape-pedicel joints, and at the joints of segments on the palps. Campaniform sensilla are concentrated as a ring around the distal margin of the pedicel, and are also scattered singly on the scape, pedicel, and flagellar segments of the antennae, and on the first segment of the maxillary palps. Occasionally, a few sensilla coeloconica and cold receptor sensilla are found on the antennal flagellum.