Postegression Feeding Enhances Growth, Survival, and Nutrient Acquisition in the Endoparasitoid Toxoneuron nigriceps (Hymenoptera: Braconidae).

Toxoneuron nigriceps Viereck (Hymenoptera: Braconidae), a koinobiont endoparasitoid of the tobacco budworm, Heliothis virescens F. (Lepidoptera: Noctuidae), derives nutrition from the host hemolymph during the internal portion of its larval development but feeds destructively on host tissues externally after egression. To investigate the importance of this tissue-feeding phase, and to evaluate the behaviors associated with postegression feeding, T. nigriceps larvae were subjected to one of four treatments: 1) allowed to carry out normal tissue feeding, 2) deprived of tissue feeding, 3) presented with tissues scraped away from the host remains, and 4) fed tissues scraped from an unparasitized H. virescens larva. Additionally, total carbohydrates, lipids, and proteins were quantified from pre and posttissue feeding T. nigriceps larvae to examine the effect of postegression feeding on parasitoid nutritional physiology. Parasitoids that received no tissues after egression, or that received tissue from an unparasitized H. virescens larva, had significantly smaller body masses at all stages than those allowed to feed naturally or fed tissues scraped from a parasitized host. Parasitoids that underwent normal host feeding after egression also reached larger masses then those fed scraped host tissue. Parasitoids that received no tissue after egression survived to adulthood significantly less often than those that were presented with any H. virescens tissue. This suggests that postegression tissue feeding is a vital developmental step for T. nigriceps, and that T. nigriceps will not only feed when normal postegression behavior is disrupted, but will also feed on unparasitized tissue. The quantification of macronutrients in the tissues of pre and posttissue feeding T. nigriceps larvae showed significantly elevated proportions of proteins, lipids, and carbohydrates in the tissues of larvae that had completed feeding, with the greatest difference being in total lipids.

The importance of odor in nest site selection by a lodger bee, Centris Bicornuta Mocsáry (Hymenoptera: Apidae) in the dry forest of Costa Rica.

The more common lodger bee occurring in the dry forest of Costa Rica, Centris bicornuta Muscáry), has been observed nesting in new nest cavities drilled into wooden blocks placed next to cavities used by another female within 2-3 days. In contrast, new nest cavities placed in similar areas with no nesting Centris nearby were not used for weeks. These observations suggest that the presence of nesting bees may play a role in nest site selection. To confirm our observations, new nest cavities were placed in areas with or without nesting. We found nest initiation in newly placed nest cavities only in areas where bees were actively nesting. To examine the possibility that nesting locations are not unique, we placed new nest cavities in new locations either with (a) a number of completed nest cavities or (b) placed alone. Within three days we only found bees nesting in the newly placed nest cavities in situation "a". The results suggested that odor might be involved. We next compared nesting in new cavities placed alone with cavities contaminated with either (a) nest entrance plug material, (b) nest nectar, (c) nest pollen or (d) a combination of pollen and nectar. Nesting was significantly low in cavities placed next to cavities with nest entrance plug material (a), and high in cavities placed next to cavities "b, c, or d". The results suggest that pollen and /or nectar odor play a role in the location of potential nest sites.

Oocyte membrane localization of vitellogenin receptor coincides with queen flying age, and receptor silencing by RNAi disrupts egg formation in fire ant virgin queens.

In ant species in which mating flights are a strategic life-history trait for dispersal and reproduction, maturation of virgin queens occurs. However, the specific molecular mechanisms that mark this transition and the effectors that control premating ovarian growth are unknown. The vitellogenin receptor (VgR) is responsible for vitellogenin uptake during egg formation in insects. In the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), virgin queens have more abundant VgR transcripts than newly mated queens, but limited egg formation. To elucidate whether the transition to egg production involved changes in VgR expression, we investigated both virgin and mated queens. In both queens, western blot analysis showed an ovary-specific VgR band (approximately 202 kDa), and immunofluorescence analysis of ovaries detected differential VgR localization in early- and late-stage oocytes. However, the VgR signal was much lower in virgin queens ready to fly than in mated queens 8 h post mating flight. In virgin queens, the receptor signal was first observed at the oocyte membrane beginning at day 12 post emergence, coinciding with the 2 weeks of maturation required before a mating flight. Thus, the membrane localization of VgR appears to be a potential marker for queen mating readiness. Silencing of the receptor in virgin queens through RNA interference abolished egg formation, demonstrating that VgR is involved in fire ant ovary development pre mating. To our knowledge, this is the first report of RNA interference in any ant species and the first report of silencing of a hymenopteran VgR.

Host regulation and release of parasitism-specific proteins in the system Toxoneuron nigriceps-Heliothis virescens.

The braconid wasp Toxoneuron nigriceps induced qualitative and quantitative changes in the protein composition of the moth Heliothis virescens host hemolymph. Total protein concentration was found to be higher in parasitized host 4 days after parasitism as compared to control hosts, mainly due to changes in a particular group of proteins. Host proteins with a molecular mass of 173 and 72 kDa were found in higher levels in the hemolymph of parasitized larvae as control hosts approached pupation, while an 80 kDa peptide was found in reduced concentration in the hemolymph of parasitized hosts. Levels of these three peptides were maintained throughout parasitoid development, while two of them (173 and 72 kDa) were cleared from the host hemolymph close to pupation. Besides the regulation of host proteins, three parasitism-specific proteins (PSPs) were released into the host hemolymph. Two of them (PSP1-MW=116 kDa, pI=6.3; PSP2-MW=114 kDa, pI=6.2) first appeared in the hemolymph of parasitized hosts soon after pupation of control host and increased in concentration as the parasitoid developed. The third PSP (PSP3-MW=56 kDa, pI=5.8) was produced towards the end of parasitoid larval development, close to parasitoid egression. Database searches based on the amino acid composition and amino terminal sequence of PSP1 and PSP2 did not produce any significant matches, while PSP3 was identified as a putative chitinase. Incubation of host derived tissues, parasitoid larvae and teratocytes in 35S conditioned media suggested PSPs were a product of teratocytes. The role of the regulation of host proteins and release of PSPs by teratocytes for the successful development of T. nigriceps are discussed.

Differential gene expression during wing morph differentiation of the ectoparasitoid Melittobia digitata (Hym., Eulophidae).

Melittobia digitata is an ectoparasitoid of solitary bees and wasps that displays a trade-off between reproduction and dispersion through the development of two wing morphs (long and short wing morphs (LWM and SWM)). The morph differentiation of this species is an exceptional adaptation to maximize host exploitation and habitat colonization, and an understanding of the mechanisms underlying this developmental process will shed light on how nutrients or environmental elicitors alter regulatory pathways leading to physiological and metabolic changes resulting in such drastic developmental rearrangements. Here we describe the differential gene expression between SWM and LWM larvae of M. digitata in order to unravel the molecular mechanisms controlling the morph differentiation in this minute parasitoid and pinpoint the pathways involved in the regulation of this developmental process. The suppression subtractive hybridization (SSH) methodology was used to isolate differentially expressed genes using mRNA populations collected soon after morph development commitment. Dot blot analysis of 384 clones from a forward SSH library identified approximately 200 differentially expressed clones, including those transcripts present in very low abundance. Further DNA sequence analysis of a sub-population of 42 clones revealed 31 putatively unique transcripts, from which 5 were further analyzed by Northern blot analysis and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The complete cDNA of one of these transcripts, a putative metalloprotease, was fully sequenced and is described. The role of the putative differentially expressed genes during the wing morph differentiation of M. digitata is discussed.

Characterization of vitellogenin in the red imported fire ant, Solenopsis invicta (Hymenoptera: Apocrita: Formicidae).

Vitellin (VN) and vitellogenin (VG) profiles were analyzed in monogyne and polygyne colonies of the red imported fire ant, Solenopsis invicta. Non-denaturing and SDS-polyacrylamide gel electrophoresis (PAGE) analyses indicated that the native VN was likely 350 kDa and comprised of two subunits in the molecular size range of 170-185 kDa. SDS-PAGE of hemolymph showed that the relative mobilities and subunit patterns of VG and VN were similar. VG was present in the hemolymph of reproductive queens; alate, virgin queens; and workers, but not in males. Anti-VN, prepared from polygyne egg homogenates, reacted with egg homogenates and with hemolymph VG from reproductive, monogyne and polygyne queens and alate, virgin polygyne queens. Analysis of circulating VG and ovarian development in alate, virgin queens showed that low levels of VG appeared by five days following adult eclosion, but egg development was not observed until seven weeks. VG was evident in newly inseminated queens, and increased steadily for the first three weeks following dealation. VG levels declined slightly near eclosion of the first workers (= nanitics) and dropped sharply after nanitic emergence at five weeks following dealation. Oocyte maturation peaked at days 15-25 following dealation, but otherwise remained low but steady. These studies provide the basis for future investigations into endocrine regulations of vitellogenesis in S. invicta queens.

Estimating parasitoid immature mortality by comparing oviposition and pupal development of trichogramma galloi Zucchi and T. pretiosum riley on natural and factitious hosts

Mortality during the immature development of T. galloi and T. pretiosum was estimated on UV-killed and live eggs of a factitious and a natural host, respectively. A staining technique was used to determine the actual parasitization of UV-treated eggs and was compared with the number of parasitoids that emerged per host egg (detectable parasitization). Effects of temperature as a factor of mortality during the immature development of both parasitoids on the factitious host was also assessed. The actual and detectable parasitization of live hosts was measured by recording both the parasitization behavior and the number of eggs where a parasitoid developed successfully. Our data show that mortality during immature development of both parasitoids may occur in live eggs of the natural host. No such mortality was observed when parasitoids developed on UV-killed eggs of the factitious host. Possible causes of parasitoid immature mortality and the effects of using UV-treated eggs of factitious hosts in estimating the parasitism capacity of Trichogramma in field conditions are discussed

Estimating parasitoid immature mortality by comparing oviposition and pupal development of Trichogramma galloi Zucchi and T. pretiosum Riley on natural and factitious hosts.

Mortality during the immature development of T. galloi and T. pretiosum was estimated on UV-killed and live eggs of a factitious and a natural host, respectively. A staining technique was used to determine the actual parasitization of UV-treated eggs and was compared with the number of parasitoids that emerged per host egg (detectable parasitization). Effects of temperature as a factor of mortality during the immature development of both parasitoids on the factitious host was also assessed. The actual and detectable parasitization of live hosts was measured by recording both the parasitization behavior and the number of eggs where a parasitoid developed successfully. Our data show that mortality during immature development of both parasitoids may occur in live eggs of the natural host. No such mortality was observed when parasitoids developed on UV-killed eggs of the factitious host. Possible causes of parasitoid immature mortality and the effects of using UV-treated eggs of factitious hosts in estimating the parasitism capacity of Trichogramma in field conditions are discussed.

A possible mechanism for the physiological suppression of conspecific eggs and larvae following superparasitism by solitary endoparasitoids.

Competition for possession of a host by internal solitary parasitoids has been attributed to physical combat and physiological suppression, but the mechanisms that result in what has been referred to as physiological suppression is poorly understood. Some insights are provided by the studies reported here using the solitary endoparasitoid, Campoletis sonorensis (Cameron). Embryos of C. sonorensis less than ten hours old rarely hatch in various artificial media, while embryos twenty hours or older generally hatch. These results suggest that young embryos in which the embryonic membranes have not yet formed are only able to develop in a narrow range of environments represented by the nonparasited hemolymph. In contrast, embryos in which the embryonic membranes are formed are able to develop in a wide range of environments represented by parasitized hemolymph which has been shown by a number of studies to change. These ideas were given support by studies reported here, where young and older eggs were incubated singly or paired. We suggest the general changes in the hemolymph of a parasitized host become unfavorable for the development of newly oviposited eggs.

The in vitro development from egg to prepupa of Campoletis sonorensis (Hymenoptera: Ichneumonidae) in an artificial medium: importance of physical factors.

A further attempt to improve the in vitro rearing of the solitary larval endoparasitoid Campoletis sonorensis (Hymenoptera: Ichneumonidae) was made after third instars were obtained in previous studies. Respiration was an important factor for the parasitoid's continued development: both small diet volumes that exposed the larval cuticle to air and O(2)greatly improved growth and development of C. sonorensis. Fifth instars were obtained when the parasitoid larvae were reared in medium volumes of 0.25ml and 0.1ml per well. Increasing the O(2) tension of the air resulted in larger larvae and prepupae. The agar layer under the nutritional medium was designed to slowly remove water from the diet. The results indicated that a drier environment was important for the parasitoid at the terminal stage. One percent and 0.8% agar gels, which removed water from the diet more rapidly, provided a drier environment and resulted in improved larval growth compared to the 0.6% agar gel. The detrimental effect of higher concentration of chicken egg yolk mixed in the diet was eliminated by using a dry egg yolk layer covered by an agar layer. This sandwich technique resulted in more parasitoid larvae molting to the final larval instar (5th) and the formation of many prepupae. However, the development of prepupae was premature and none of the fifth instar larvae successfully formed a normal cocoon.

Diversity in cyclic sesquiterpene production by Gossypium hirsutum.

Major sesquiterpene components of oil of Texas Race Stock 810 of Gossypium hirsutum were alpha- and beta-selinene. This is the seventh cyclic terpene type found to date in this genus. Both alpha- and beta-selinene, along with aromadendrene, were found but only as minor components of extracts of several domestic cultivars of G. hirsutum.

Alkadienes mediating courtship in the parasitoidCardiochiles nigriceps (Hymenoptera: Braconidae).

Courtship inCardiochiles nigriceps (Viereck) (Hymenoptera: Braconidae) involves male attraction to females and male antennation of females followed by mounting and copulation. Once the female is located, antennation and mounting of the female are mediated by the hydrocarbon fraction of female's Dufour's gland and cuticle. The identification of the cuticular hydrocarbons of males and females revealed a mixture of alkanes and alkenes, in addition to a series of alkadienes specific to females. These female-specific alkadienes reported from Braconidae are unusual among insect alkadienes in that the second double bond occurs in the middle of the molecule. Bioassays with three available alkadienes revealed that contact behaviors (antennation and mounting) are in part mediated by the (Z,Z)-7, 13-heptacosadiene and at least one other alkadiene in combination with other hydrocarbons found in males and females.

A long-range attractant kairomone for egg parasitoidTrissolcus basalis, isolated from defensive secretion of its host,Nezara viridula.

A short-chain α,ß-unsaturated aldehyde, (E)-2-decenal, present in the defensive metathoracic gland ofNezara viridula (L.) (Heteroptera: Pentatomidae), stimulates a behavioral response in the egg parasitoidTrissolcus basalis (Woll.) (Hymenoptera: Scelionidae). Preliminary studies showed thatT. basalis are attracted to an area containing adultN. viridula, but we also found that femaleT. basalis would examine and probe glass beads coated with an acetone extract of the metathoracic gland from males or females. Using this bioassay, the kairomone was isolated by bioassay directed by preparative gas chromatography and identified by NMR and mass spectrometry as (E)-2-decenal. The biological activity of the identified aldehyde was compared with analogs to determine specificity. An unstable Z isomer was found to be more active but not present in detectable or behaviorly relevant levels in the host, based on the bell-shaped dose-response curve of the two isomers. An investigation was also designed to determine if theE isomer was also responsible for the egg recognition kairomone activity previously reported. However, no 2-decenal isomers were detected in host egg extracts and the chemical characteristics of the 2-decenal isomers differ from the unidentified egg recognition kairomone. The role of the (E)-2-decenal in attracting femaleT. basalts toN. viridula was demonstrated in a Y-tube olfactometer; this alk-2-enal appears to act as a long-range kairomone orientingT. basalis toNezara populations.

Susceptibility and behavioral response of red imported fire ant (Hymenoptera: Formicidae) to selected entomogenous nematodes (Rhabditida: Steinernematidae & Heterorhabditidae).

Pathogenicity of infective juveniles of selected Steinernema spp. and Heterorhabditis spp. toward developing and reproductive stages of the red imported fire ant, Solenopsis invicta Buren, was tested under laboratory conditions. At 10(3)-10(5) infective juveniles per Petri dish, mortality of reproductive larvae, pupae, and alates ranged from 28 to 100% at higher doses after 96 h at 23-25 degrees C. Steinernema carpocapsae All was the most consistent species tested; this nematode caused mortality of fire ant larvae, pupae, and alates of 82-94, 64-96, and 38-99%, respectively. Although not susceptible to nematode infection, worker ants vigorously preened nematodes from brood, alates, and themselves. In a field study, S. carpocapsae (5 x 10(6) and 2 x 10(6) drench, 2 x 10(6) infective juvenile infection) was applied to active fire ant mounds in 3.8-liter suspensions. Hydramethylnon (75 ml), a water drench, a water injection, and untreated fire ant mounds were marked and treated. Overall activity in mounds treated with nematodes of hydramethylnon ranged from 40 to 48%. Satellite mound activity accounted for 32-44% of overall activity in mounds treated with nematodes 2 wk after treatment. However, 6 wk after treatment, activity in mounds treated with hydramethylnon was 44%; activity of mounds treated with nematodes ranged from 52 to 80%. Satellite mound activity accounted for 0-24% of overall activity. Whereas a soil drench of S. carpocapsae showed potential as a control method for the red imported fire ant, colony relocation after nematode treatment could limit overall efficacy unless application techniques are developed to overcome or take advantage of the movement.

Distribution and density of polygyne fire ants (Hymenoptera: Formicidae) in Texas.

Multiple-queen or "polygyne" Solenopsis invicta Buren colonies are a serious economic and environmental concern because they occur in much higher densities than the monogyne form. Polygyne colonies have been found at numerous locations in the United States; nevertheless, the frequency and distribution of this form are poorly known. Almost 700 roadside sites in 168 Texas counties were surveyed. Polygyny was discovered at 54% of the infested sites. Polygyne populations were scattered in a mosaic across Texas. The frequency of polygyny varied somewhat with geographic region, but the pattern was generally unrelated to habitat and environmental conditions. Polygyne sites averaged more than twice as many mounds per hectare as monogyne sites. Populations of monogyne and polygyne forms were slightly lower in cooler and drier portions of the state. Mounds of both forms were about the same size. Polygyny was correlated with lower rates of sexual production and reduced numbers of native ants. The high frequency of polygyny in Texas indicates that the fire ant problem in the state is much greater than previously realized.

Evidence for a two-component external marking pheromone system in an aphid hyperparasitoid.

The megaspilidDendrocerus carpenteri (Curtis) is a solitary ectohyperparasitoid of aphid primary parasitoids. It avoids superparasitism, but the avoidance rate depends on the amount of time elapsed after the first oviposition. In addition, females discriminate in a time-dependent manner between hosts parasitized by themselves and hosts parasitized by conspecifics. They recognize parasitized hosts by means of two externally perceivable chemical markers that are deposited on the mummy shell. One of the markers is highly active, short-lived, not passable through gas chromatography (GC), and freezing sensitive; the other is moderately active, persistent, GC-passable and freezing tolerant. Using two markers is believed to be advantageous for the hyperparasitoid female, as their ratio provides information on mark age and on the producer of the mark (the female herself or a conspecific female).

Change in mandibular and mesosomal gland contents of maleXylocopa micans (Hymenoptera: Anthophoridae) associated with mating system.

Males ofXylocopa micans employ two mating systems in south-central Texas. They defend territories around flowering wisteria and redbud in March and April, and they maintain nonresource-based or landmark territories in July and August. Mandibular and mesosomal gland contents (analyzed by GC-MS) are different in bees employing the two mating systems. Mandibular glands contain only straight-chain hydrocarbons in bees defending floral resources, but include ethyl oleate in bees defending landmark territories. Mesosomal glands contain saturated, mono- and diunsaturated straight-chain hydrocarbons and methyl and ethyl esters of long-chain fatty acids. The major ethyl ester, ethyl oleate, comprises only 1.1% of mesosomal gland contents in bees defending floral resources but comprises 39.7% of gland content of bees defending landmark territories. These findings are discussed relative to the proposed sex pheromone function ofXylocopa gland secretions.

Electroantennogram responses ofCampoletis sonorensis (hymenoptera: Ichneumonidae) to chemicals in cotton (Gossypium hirsutum L.).

Combined gas chromatography-electroantennogram (GC-EAG) recording ofCampoletis sonorensis (Cameron) responses to cotton plant volatile chemicals was performed.C. sonorensis antennal olfactory receptors respond differentially to green leaf, mono-, and sesquiterpene chemicals that have been identified previously in cotton. EAG depolarizations to green leaf chemicals were greater than to terpenes.

Collection of pheromone from atmosphere surrounding boll weevils,Anthonomus grandis.

An effluvial method was developed to collect the pheromone, grandlure from actively calling male boll weevils,Anthonomus grandis Boheman. The adsorbant, Porapak Q (ethylvinylbenzene-divinylbenzene), was utilized to trap and concentrate the pheromone. Captured pheromone was desorbed from columns packed with Porapak Q by elution withn-pentane and quantified by capillary column gas-liquid chromatography. In recovery studies with known amounts of synthetic grandlure, we found that the amount of each pheromone component collected was a function of collection duration, elution volume, and initial concentration. This effluvial method was capable of recovering as much as 94.9% of a known quantity (80 µg) of grandlure. The chromatograms were free of extraneous peaks. In studies of insect-produced pheromone, the effluvial method was used to collect pheromone from the air space surrounding male boll weevils as they fed on flower buds from CAMD-E cotton. The quantity and quality of boll-weevil-produced pheromone was determined for days 6, 8, 10, 11, 12, 13, and 14 of boll weevil adulthood. The maximum quantity of natural pheromone was produced on day 13 (4.2 µg/weevil) with a pheromone component ratio of 2.41∶2.29∶0.95∶1 for components I, II, III, and IV, respectively. The effluvial method described in this report is an efficient method to collect and quantify boll weevil pheromone from the atmosphere surrounding actively calling insects. Other applications of this method are suggested.

Kairomonal activities of 2-acylcyclohexane-1,3 diones produced byEphestia kuehniella zeller in eliciting searching behavior by the parasitoidBracon hebetor (say).

2-Acylcyclohexane-1,3-diones produced in the mandibular glands ofEphestia kuehniella Zeller (Lepidoptera: Pyralidae) fifth instars acted as arrestment and host-trail following kairomones for the parasitoid,Bracon hebetor (Say) (Hymenoptera: Braconidae). The behavioral response of the parasitoids to the kairomone varied with kairomone concentration and distribution. However, only small differences in activity were noted for the different mandibular gland components.B. hebetor females that encountered filter paper patches impregnated with the kairomone exhibited antennation and probing behavior. Females followed trails formed with 2-[(Z,E)12,14-hexadecadienoyl]cyclohexane-1,3-dione in the same manner exhibited with host-made trails. At concentrations of 1.0 µg/mm and above, trail following was interrupted by frequent probing.