Ovipositional Behavior of the Egg Parasitoid Gryon pennsylvanicum (Hymenoptera: Scelionidae) on Two Squash Bug Species Anasa tristis (Hemiptera: Coreidae) and Anasa armigera: Effects of Parasitoid Density, Nutrition, and Host Egg Chorion on Parasitism Rates.

This study examined the ovipositional behavior of Gryon pennsylvanicum Ashmead (Hymenoptera: Scelionidae) on egg masses of two squash bug species Anasa tristis DeGeer and Anasa armigera Say (Hemiptera: Coreidae) by evaluating how parasitoid density and access to nutrition influenced percent parasitism on egg masses of different sizes in laboratory tests. When three parasitoids were exposed to A. tristis egg masses with only three to five eggs, 72.7% of parasitoids became trapped in the eggs and failed to emerge successfully. These results suggest that competition between larvae within the egg may have reduced the fitness of the surviving parasitoid. Continual access to honey water did not significantly influence parasitism rates on A. armigera egg masses and only increased parasitism on A. tristis egg masses with 20-25 eggs. Overall, parasitism rates were higher on A. armigera egg masses than on A. tristis egg masses, and parasitoids were more likely to emerge successfully from A. armigera eggs than from A. tristis eggs. Parasitoids spent the same amount of time probing eggs of the two species, but they spent significantly more time drilling into A. tristis eggs than A. armigera eggs. Measurements taken using transmission electron microscopy determined that the average combined width of the epicuticle and exocuticle of the egg chorion was significantly greater for A. tristis eggs than for A. armigera eggs. This difference may account for the lower rates of parasitism and parasitoid emergence and for the increased time spent drilling into A. tristis eggs compared with A. armigera eggs.

Comparative Study of Egg Parasitism by Gryon pennsylvanicum (Hymenoptera: Scelionidae) on Two Squash Bug Species Anasa tristis and Anasa armigera (Hemiptera: Coreidae).

This study evaluated how the size of the egg mass and the parasitoids prior exposure to eggs influenced parasitism rates by Gryon pennsylvanicum Ashmead (Hymenoptera: Scelionidae) on egg masses of two squash bug species, Anasa tristis DeGeer and Anasa armigera Say (Hemiptera: Coreidae). G. pennsylvanicum is the primary egg parasitoid of A. tristis. There were no published reports available on egg parasitism of A. armigera. In choice tests, there was no difference in host acceptance by G. pennsylvanicum of egg masses of the two squash bug species. In no-choice tests, overall parasitism rates were significantly higher on A. armigera egg masses than on A. tristis egg masses. Naive parasitoids had significantly higher parasitism rates than experienced parasitoids on egg masses of both squash bug species. In a comparison of parasitism rates of field-collected and laboratory-tested A. tristis egg masses of different sizes, parasitism rates were similar in the field and in the laboratory, with the exception of egg masses with > 25 eggs. Only 17.9% of eggs were parasitized in the laboratory, compared with 36.4% in the field. Results of this study indicate that transient egg limitation prevents G. pennsylvanicum from ovipositing in every available host egg in large squash bug egg masses. The low parasitism rate of G. pennsylvanicum on large egg masses may limit its effectiveness as a biological control agent of squash bugs.

Removing external DNA contamination from arthropod predators destined for molecular gut-content analysis.

Ecological research requires large samples for statistical validity, typically hundreds or thousands of individuals, which are most efficiently gathered by mass-collecting techniques. For the study of interspecific interactions, molecular gut-content analysis enables detection of arthropod predation with minimal disruption of community interactions. Field experiments have demonstrated that standard mass-collection methods, such as sweep netting, vacuum sampling and foliage beating, sometimes lead to contamination of predators with nontarget DNA, thereby compromising resultant gut-content data. We deliberately contaminated immature Coleomegilla maculata and Podisus maculiventris that had been fed larvae of Leptinotarsa decemlineata by topically applying homogenate of the alternate prey Leptinotarsa juncta. We then attempted to remove contaminating DNA by washing in ethanol or bleach. A 40-min wash with end-over-end rotation in 80% EtOH did not reliably reduce external DNA contamination. Identical treatment with 2.5% commercial bleach removed most externally contaminating DNA without affecting the detectability of the target prey DNA in the gut. Use of this bleaching protocol, perhaps with minor modifications tailored to different predator-prey systems, should reliably eliminate external DNA contamination, thereby alleviating concerns about this possible source of cross-contamination for mass-collected arthropod predators destined for molecular gut-content analysis.

Insecticidal activity of some reducing sugars against the sweet potato whitefly, Bemisia tabaci, Biotype B.

The effects of 16 sugars (arabinose, cellobiose, fructose, galactose, gentiobiose, glucose, inositol, lactose, maltose, mannitol (a sugar alcohol), mannose, melibiose, ribose, sorbitol, trehalose, and xylose) on sweet potato whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) survival were determined using in vitro bioassays. Of these sugars, arabinose, mannose, ribose, and xylose were strongly inhibitory to both nymphal and adult survival. When 10% mannose was added to the nymphal diet, 10.5%, 1.0%, and 0% developed to the 2nd, 3rd, and 4th instars, respectively. When 10% arabinose was added, 10.8% and 0% of the nymphs molted to the 2nd and 3rd instars, respectively. Addition of 10% xylose or ribose completely terminated B. tabaci development, preventing the molt to the 2(nd) instar. With decreasing sugar concentrations the inhibitory effect was significantly reduced. In tests using adults, arabinose, galactose, inositol, lactose, maltose, mannitol, mannose, melibiose, ribose, sorbitol, trehalose, and xylose significantly reduced mean day survival. Mortality rates were highest when arabinose, mannitol, mannose, ribose, or xylose was added to the diet. Mean day survival was less than 2 days when adults were fed on diet containing 10% of any one of these five sugars. When lower concentrations of sugars were used there was a decrease in mortality. Mode of action studies revealed that toxicity was not due to the inhibition of alpha glucosidase (converts sucrose to glucose and fructose) and/or trehalulose synthase (converts sucrose to trehalulose) activity. The result of agarose gel electrophoresis of RT-PCR products of bacterial endosymbionts amplified from RNA isolated from whiteflies fed with 10% arabinose, mannose, or xylose indicated that the concentration of endosymbionts in mycetomes was not affected by the toxic sugars. Experiments in which B. tabaci were fed on diets that contained radio-labeled sucrose, methionine or inulin and one or none (control) of the highly toxic sugars showed that radioactivity (expressed in DPM) in the body, in excreted honeydew and/or carbon dioxide, was significantly reduced as compared to controls. Thus, it appears that the ability of insecticidal sugars to act as antifeedants is responsible for their toxicity to B. tabaci.

Critical feeding periods for last instar nymphal and pharate adults of the whiteflies, Trialeurodes vaporariorum and Bemisia tabaci.

A critical feeding period is the time after which 50% of a given species of insect can be removed from its food source and complete development by undergoing adult eclosion. The critical feeding period was determined for the greenhouse white fly, Trialeurodes vaporariorum, and the sweet potato whitefly, Bemisia tabaci (Biotype B) (Homptera/Hemiptera: Aleyrodidae). Fourth (last) instar and pharate adult whiteflies were removed from green bean leaves, staged, placed on filter paper in small Petri dishes containing drops of water, and observed daily for eclosion. For T. vaporariorum reared at 25 degrees C and L:D 16:8, 55 and 80% adult eclosion were observed when whiteflies were removed at stages 4 (0.23-0.26 mm in body depth) and 5 (> or = 0.27 mm in body depth), respectively, so that at least 50% eclosion was only achieved in this species of whitefly when adult eye development had already been initiated (in Stage 4), and 80% eclosion when adult wing development had been initiated (Stage 5). In contrast, 63% of B. tabaci emerged as adults if removed from the leaf at Stage 3 (0.18-0.22 mm in body depth), and 80% emerged if removed at Stage 4/5, stages in which adult formation had not yet been initiated. The mean number of eggs laid by experimental (those removed at Stages 4-5, 6-7 or 8-9) and control (those that remained on the leaf prior to eclosion) whiteflies, and the mean percent hatch of these eggs were not significantly different in experimental and control groups. Stages 7, 8 and 9 are characterized by a light red adult eye, medium red bipartite adult eye and dark red or red-black bipartite adult eye, respectively. Mean adult longevity also was not significantly different between experimental and control groups. However, for all groups of T. vaporariorum, adult female longevity was significantly (at least 2 times) greater than male longevity. Our results identify the critical feeding periods for last instar/pharate adults of two important pest species of whitefly. Since in both species of whitefly the critical feeding period is achieved when weight gain reaches a plateau, it appears that the critical feeding period is more closely correlated with the attainment of a critical weight than with either the time that ecdsyteroid titers first peak or the time when adult development is initiated.

Host-parasite interactions between whiteflies and their parasitoids.

There is relatively little information available concerning the physiological and biochemical interactions between whiteflies and their parasitoids. In this report, we describe interactions between aphelinid parasitoids and their aleyrodid hosts that we have observed in four host-parasite systems: Bemisia tabaci/Encarsia formosa, Trialeurodes vaporariorum/E. formosa, B. tabaci/Eretmocerus mundus, and T. lauri/Encarsia scapeata. In the absence of reported polydnavirus and teratocytes, these parasitoids probably inject and/or produce compounds that interfere with the host immune response and also manipulate host development to suit their own needs. In addition, parasitoids must coordinate their own development with that of their host. Although eggs are deposited under all four instars of B. tabaci, Eretmocerus larvae only penetrate 4th instar B. tabaci nymphs. A pre-penetrating E. mundus first instar was capable of inducing permanent developmental arrest in its host, and upon penetration stimulated its host to produce a capsule (epidermal in origin) in which the parasitoid larva developed. T. vaporariorum and B. tabaci parasitized by E. formosa initiated adult development, and, on occasion, produced abnormal adult wings and eyes. In these systems, the site of parasitoid oviposition depended on the host species, occurring within or pressing into the ventral ganglion in T. vaporariorum and at various locations in B. tabaci. E. formosa's final larval molt is cued by the initiation of adult development in its host. In the T. lauri-E. scapeata system, both the host whitefly and the female parasitoid diapause during most of the year, i.e., from June until the middle of February (T. lauri) or from May until the end of December (E. scapeata). It appears that the growth and development of the insects are directed by the appearance of new, young foliage on Arbutus andrachne, the host tree. When adult female parasitoids emerged in the spring, they laid unfertilized male-producing eggs in whiteflies containing a female parasitoid [autoparasitism (development of male larvae utilizing female parasitoid immatures for nutrition)]. Upon hatching, these male larvae did not diapause, but initiated development, and the adult males that emerged several weeks later mated with available females to produce the next generation of parasitoid females. Thus, the interactions that exist between whiteflies and their parasitoids are complex and can be quite diverse in the various host-parasitoid systems.

Identification of the molting hormone of the sweet potato (Bemisia tabaci) and greenhouse (Trialeurodes vaporariorum) whitefly.

In order to identify the whitefly molting hormone, whole body extracts of mature 4th instar and newly formed pharate adult Bemisia tabaci (Biotype B) and Trialeurodes vaporariorum were prepared and subjected to reverse phase high performance liquid chromatography (RPHPLC). Ecdysteroid content of fractions was determined by enzymeimmunoassay (EIA). The only detectable ecdysteroids that were present in significant amounts in whitefly extracts were ecdysone and 20-hydroxyecdysone. The concentrations of 20-hydroxyecdysone in B. tabaci and T. vaporariorum extracts, respectively, were 40 and 15 times greater than the concentrations of ecdysone. The identity of the two ecdysteroids was confirmed by normal phase high performance liquid chromatography (NPHPLC). When ecdysteroid content of RPHPLC fractions was assayed by radioimmunoassay (RIA), small amounts of polar ecdysteroids were also detected indicating that these ecdysteroids have a very low affinity for the antiserum used in the EIA. Ecdysteroid at 10.4 mM administered by feeding stimulated 2nd instar whitefly nymphs to molt. Based on our results, it appears that 20-hydroxyecdysone is the whitefly molting hormone.

The broadly insecticidal Photorhabdus luminescens toxin complex a (Tca): activity against the Colorado potato beetle, Leptinotarsa decemlineata, and sweet potato whitefly, Bemisia tabaci.

Toxin complex a (Tca), a high molecular weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens, has been found to be orally toxic to both the Colorado potato beetle, Leptinotarsa decemlineata, and the sweet potato whitefly, Bemisia tabaci biotype B. The 48 hour LC50 for Tca against neonate L. decemlineata was found to be 2.7 ppm, and the growth of 2nd instar L. decemlineata exposed to Tca for 72 hours was almost entirely inhibited at concentrations above 0.5 ppm. B. tabaci was highly susceptible to Tca as well; newly emerged nymphs that were artificially fed Tca developed poorly, or not at all. Tca concentrations between 0.1 and 0.2 ppm reduced the number of nymphs reaching the second instar by 50%. In addition, a preparation of Tca missing two prominent subunits, TcaAii and TcaAiii, was found to be at least as toxic to L. decemlineata and B. tabaci as Tca itself, indicating that the activity of Tca is not dependant on the presence of these subunits at the time of ingestion.

Age-specific interaction between the parasitoid, Encarsia formosa and its host, the silverleaf whitefly, Bemisia tabaci (Strain B).

The effect of hostage, the instar of Bemisia tabaci (Gennadius) parasitized, on the growth and development of Encarsia formosa (Gahan) was studied. E. formosa was able to parasitize and complete its life cycle no matter which instar of B. tabaci (Strain B), [also identified as B. argentifolii (Bellows and Perring)], was provided for oviposition, but parasitoid development was significantly slower when 1st or 2nd instar B. tabaci rather than 3rd or 4th instars were parasitized. Host age influenced the day on which E. formosa nymphs hatching from eggs was first observed. Mean embryonic development was significantly longer when 1st (5.4 days) rather than 2nd, 3rd or 4th instars (4.1, 3.4 and 3.5 days, respectively) were parasitized. The duration of the 1st instar parasitoid and the pupa, but not the 2nd or 3rd instar parasitoid, were also significantly greater when 1st instars were parasitized than when older host instars were parasitized. Interestingly, no matter which instar was parasitized, the parasitoid did not molt to the 3rd instar until the 4th instar host had reached a depth of about 0.23 mm (Stage 4-5) and had initiated the nymphal-adult molt and adult development. Histological studies revealed that whitefly eye and wing structures had either disintegrated or were adult in nature whenever a 3rd instar parasitoid was present. It appears, then, that the molt of the parasitoid to its last instar is associated with the host whitefly's nymphal-adult molt. However, the initiation of the host's final molt, while a prerequisite for the parasitoid's 2nd-3rd instar molt, did not necessarily trigger this molt. In contrast to its significant effect on various aspects of parasitoid development, host instar did not significantly influence the mean size of the parasitoid larva, pupa, or adult. Larval and pupal length and adult head width were similar for all parasitoids, regardless of which host instar was parasitized as was adult longevity. Adult parasitoid emergence was more synchronous when 2nd, 3rd and 4th instars were parasitized than when 1st instars were parasitized. Results are compared with those reported when the greenhouse whitefly, Trialeurodes vaporariorum, was parasitized by E. formosa, and provide possible explanations for why T. vaporariorum is a more suitable host than B. tabaci for E. formosa.

Co-development of Encarsia formosa (Hymenoptera: Aphelinidae) and the greenhouse whitefly, Trialeurodes vaporariorum (Homoptera: Aleyrodidae): a histological examination.

Using histological techniques, we have simultaneously examined the co-development of the Aphelinid parasitoid Encarsia formosa and its host the greenhouse whitefly, Trialeurodes vaporariorum. Previously we have determined that regardless of the whitefly instar parasitized, parasitoid larvae would not molt to their final instar until the whitefly reaches its maximum dimensions. In unparasitized T. vaporariorum, this point in development corresponds to the initiation of the adult molt. In part, this study was conducted to determine the developmental state of parasitized whiteflies at the time they achieve their maximum dimensions. It was found that parasitized final instar T. vaporariorum do, in fact, undergo a final molt and that E. formosa larvae will not molt to their final instar until this has occurred. The timing of the final whitefly molt appears unaffected by parasitization. The commonly observed melanization of parasitized whiteflies appears to be a consequence of this molt. In addition, we have discovered that the adult wasp oviposits within the ventral ganglion of the whitefly, and that major organ systems of the whitefly persist very late into parasitoid development. We also report the presence of possible endosymbiotic bacteria residing in the fatbody of E. formosa.

The nymphal-adult molt of the silverleaf whitefly (Bemisia argentifolii): Timing, regulation, and progress.

The developmental progress of silverleaf whitefly (Bemisia argentifolii) 3rd instars and 4th instar/pharate adults was monitored using a tracking system that had been designed to identify synchronous individuals in another species of whitefly, the greenhouse whitefly, Trialeurodes vaporariorum. When reared on greenbean under conditions of LD 16:8 and a temperature of 26 +/- 2 degrees C, the body depth of 3rd instar SLWFs increased from approximately 0.04 mm (Stage 2) to 0.175-0.2 mm (Stage 7-8) and the body depth of the 4th instar increased from approximately 0.1 mm (Stage 1) to 0.25-0.30 mm (Stage 4-5). The durations of the 3rd instar and the 4th instar/pharate adult were approximately 3 and 7 days, respectively. Examination of coronal sections of 4th instars revealed that adult eye and wing development are initiated during Stage 6, the stage in which an external examination showed that the eye has begun to undergo pigment diffusion. Ecdysteroid titers peaked at approximately 400 fg/ micro g protein during stages 4 through 6A of the 4th instar, i.e., just prior to and upon the initiation of the pharate adult stage. Although adult development is initiated later in the SLWF than in the GHWF (adult eye and wing development begin in Stages 4 and 5, respectively, in GHWFs), the same rapidity of metamorphosis is observed in both species. Within approximately 24 h, the simple bi-layered wing bud developed into a deeply folded wing of nearly adult proportions and within an additional 12-24 h, the nymphal eye and wing bud had been replaced by the well-differentiated eye and wing of the adult whitefly. Our study is the first to describe the regulation, timing, and progress of the nymphal-adult molt and of the structural changes that accompany nymphal-adult metamorphosis in the SLWF.

Growth and development of Encarsia formosa (Hymenoptera: Aphelinidae) in the greenhouse whitefly, Trialeurodes vaporariorum (Homoptera: Aleyrodidae): effect of host age.

The tiny parasitoid wasp, Encarsia formosa, has been used successfully to control greenhouse whiteflies (GHWFs) in greenhouses in many countries throughout the world. Therefore, there has been considerable interest in developing methods for artificially rearing this wasp. However, little information is available concerning the regulation of its development including the host-parasitoid interactions that are required for the parasitoid to complete its life cycle. Here we confirm that parasitoid developmental rates differ significantly based upon the host instar parasitized. Development was faster when 3rd and 4th instar GHWFs were offered for parasitization than when 1st or 2nd instars were used. Our results show that it is primarily the embryo and the first two parasitoid instars that exhibit prolonged developmental times when 1st and 2nd instar whiteflies are parasitized. Although percent emergence was not affected by host age at the time of parasitization, adult longevity as well as adult emergence pattern varied greatly depending upon the instar parasitized. When 3rd and 4th instar GHWFs were selected for oviposition, adult wasps lived significantly longer than when 1st or 2nd instars were used; also, there was a sharp emergence peak on the 2nd day after emergence was first observed (reduced or absent when 1st or 2nd instar GHWFs were parasitized) and the emergence period was reduced from between 8 and 11 days to 5 days. In general, the younger the host instar parasitized, the less synchronous was parasitoid development. Previous reports that E. formosa will not molt to the 2nd instar until the host has reached its 4th instar were not confirmed. When 1st instar host nymphs were parasitized, 2nd instar parasitoids were detected in 3rd instar hosts. Importantly, however, no matter which instar was parasitized, the parasitoid never molted to its last instar until the host had reached Stage 5 of its last instar, a stage in which host pharate adult formation has been initiated. It appears, then, that a condition(s) associated with host pharate adult formation is required for the parasitoid's final larval molt. Results reported here should facilitate the development of in vitro rearing systems for E. formosa.