Jing S. Hu

Timing and ecdysteroid regulation of the molt in last instar greenhouse whiteflies ( Trialeurodes vaporariorum )

A system of markers has been devised to track the development of 3rd and 4th instar/pharate adult greenhouse whiteflies. Instars were identified based on measurements of body width and body length. Depending upon the host plant, the product of the two measurements was exceptionally useful in distinguishing between instars. Body depth was used to divide the 3rd instar into eight stages and body depth and color and appearance of the developing adult eye were used to divide the 4th instar/pharate adult into nine stages. Under conditions of L:D 16:8 and a temperature of 26+/-2 degrees C, the body depth of 3rd instars reared on greenbean increased from 0.025 (stage 1) to 0.2mm (stage 8) and the instar duration was approximately 3 days. The body depth of 4th instars increased from approximately 0.1+/-0.02 (Stage 1) to 0.3+/-0.03mm (Stage 5) and then remained constant or decreased slightly during adult development. Ecdysteroid titers peaked at approximately 120fg/&mgr;g protein during Stages 3 through 6 of the 4th instar. Based on an external examination of developing 4th instars and the fluctuations in ecdysteroid titer, it appears that adult development is initiated in Stage 4 or 5 4th instars. Results from histological studies support this view. In Stage 4 nymphs, a subtle change was observed in the corneagenous cells of the eye. However, most Stage 4 4th instars possessed wing development characteristic of earlier, immature stages. In all Stage 5 insects, wing development had been initiated and the corneagenous cells had become quite distinct. In Stage 6 whiteflies, the wing buds were deeply folded and by Stage 7, spines were observed on the new cuticle, indicating that the adult cuticle was well-formed by this stage. Our study is the first to investigate the timing and regulation of the molt, to monitor ecdysteroid titers in precisely staged 4th instar whiteflies and to examine the internal anatomical changes associated with metamorphosis in these tiny homopteran insects.

Artificial diets for rearing the Colorado potato beetle, Leptinotarsa decemlineata

Abstract Colorado potato beetles have been reared successfully through 12 generations on artificial diets containing either 2.5% potato leaf powder or 2.5% lettuce leaf powder/0.75% potato leaf powder. For all but one of the treatment groups, the mean duration of each of the four larval stages was between 0.8 and 1.5 days longer than the durations exhibited by control beetles that had been fed on potato leaves. Maximum weights of prepupae, newly emerged adults and day 5 – 9 adults were approximately 78, 80 and 82%, respectively, of the weights for comparable stages of control beetles. Mean percent mortality for 1st instars was two to six times higher for artificial diet-fed CPBs than for leaf-fed beetles. However, since pupal mortality was four times higher for control beetles than for beetles reared on artificial diet, mean percent total mortality (newly hatched through the 9 day old adult) was equivalent for leaf-fed beetles and for later generations of potato and Lettuce+Potato diet-fed CPBs. Hemolymph ecdysteroid levels and fluctuations in mature 4th instar larvae and prepupae were similar in controls and experimental groups. Number of hatchlings produced per adult pair per day (fertility) was approximately eight times greater in control beetles than in later generations of artificial diet-fed beetles, primarily because fewer egg masses were laid per day, percent hatch was lower and cannibalism of eggs was higher in these latter groups. Interestingly, the mean percent hatch, although only 68% of the control value, was 1.5 times greater for beetles reared on diet containing lettuce-leaf powder, and a small percentage of potato leaf powder, than on diet containing only potato leaf powder. Percent hatch was equal for beetles fed on diet containing only lettuce-leaf powder and those fed on potato leaves. Finally, it is noteworthy that the quality of eggs, as judged by the ability of the wasp parasitoid, Edovum puttleri, to parasitize and develop in the eggs, was similar for eggs produced by control beetles and for those produced by beetles fed on potato and Lettuce+Potato diets. The diets and rearing system described here will be useful for providing beetles on a year-round basis for experiments designed to evaluate the effects of potential insect control agents, to investigate the mechanism(s) by which insects become resistant to control agents and for other applied and fundamental studies related to the control of this serious pest. The use of lettuce leaf powder in place of most of the potato leaf powder is especially advantageous because of the much reduced cost and greater availability of lettuce as compared to potato leaves.

Effects of selected physical and chemical treatments of Colorado potato beetle eggs on host acceptance and development of the parasitic wasp, Edovum puttleri.

Effects of various physical and chemical treatments of Colorado potato beetle [Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)] eggs on parasitization and development of the egg parasitoid Edovum puttleri (Hymenoptera: Eulophidae) were investigated. UV irradiation did not affect host acceptance but reduced host suitability for UV exposure times ≥90 min. Susceptibility of host eggs to UV irradiation varied with host age; eggs were most vulnerable to damage from irradiation at 12, 18, and 24 h post‐oviposition. The rate of parasitization also was influenced by host age. Percent parasitization was greatest in freshly laid eggs and 24–30 h old eggs. Seventy‐seven percent of host eggs frozen at −20 °C (5 min) were parasitized by E. puttleri, but extended exposure of eggs to −20 °C reduced both acceptance and suitability. Host eggs that had been washed with hexane (removal of kairomone and sticky layer) also were parasitized. After 5 min of washing, application of kairomone significantly increased the rate of parasitism (from 74.7% to 88.2%), but with longer periods of washing, kairomone application had no significant effect on percent parasitism. Thus, the sticky material(s) coating the egg did not appear to be essential for parasitization to occur. Our results provide effective methods and times for treating Colorado potato beetle eggs to maximize parasitization and development of E. puttleri.

In vitro rearing of Edovum puttleri, an egg parasitoid of the Colorado potato beetle – development from egg through the pupal stage

A variety of semi-defined artificial diets were developed and tested for their ability to support the in vitro development of Edovum puttleri. In the most effective diet, 2.6% of E. puttleri pupated. This diet contained high levels of hen egg yolk combined with Manduca sexta larval hemolymph, or with a mixture of M. sexta egg homogenate and larval hemolymph. Egg homogenate alone (without the addition of hemolymph) was not capable of supporting the parasitoids development. Thus, hemolymph appears to contain unidentified factor(s) important for inducing pupation of the wasp. Addition of M. sexta pupal fat body tissue extract (in place of hemolymph) also promoted pupation of E. puttleri. Gypsy moth (Lymantria dispar) larval hemolymph could not replace M. sexta larval hemolymph. Fractionation irreversibly reduced the growth-promoting effects of M. sexta larval hemolymph. However, the most effective fraction contained components whose molecular weights were ≥1000 kd. In diets that were devoid of insect materials, the best results were achieved when hen egg yolk, FreAmine, yeast extract, lactalbumin, trehalose, fetal bovine serum and bovine milk were included. This is the first report of an artificial diet for in vitro rearing an eulophid parasitoid from the egg through the pupal stage.

Ecdysteroid and free amino acid content of eggs of the Colorado potato beetle, Leptinotarsa decemlineata

In order to identify components of the Colorado potato beetle (CPB) egg that may be required by Edovum puttleri, a parasitic wasp that parasitizes the CPB egg, to complete development, ecdysteroid and free amino acid content of CPB eggs were analyzed by reversed phase high pressure liquid chromatography followed by radioimmunoassay to identify ecdysteroids. Ecdysteroid titers were relatively low (<300 pg/egg) through day 2 post-oviposition and then increased sharply, reaching concentrations >2,500 pg/egg on day 3 post-oviposition. Ecdysone (E), 20-hydroxyecdysone (20E), and polar conjugates of E were prominent ecdysteroids present in eggs sampled on days 0 and 1 post-ecdysis, and E, 20E, three peaks containing more polar ecdysteroids (metabolic inactivation products), and polar conjugates of E were present in eggs sampled on day 2. Thus, at a time when parasitization of CPB eggs by E. puttleri is relatively high (0-48 h), physiologically-active ecdysteroids (20E and perhaps E are physiologically active) are present at concentrations between 50 and 200 pg/egg. Ecdysone and 20E reached their highest levels in day-3 eggs, indicating that ecdysteroid may direct physiological processes associated with the completion of CPB embryonic development. In day-4 eggs, the concentration of E and 20E fall dramatically and polar metabolites of E and/or 20E are now responsible for the high ecdysteroid content of the eggs. Interestingly, conjugates of E decrease to relatively low levels in day-3 eggs and are absent in day-4 eggs. Therefore, it is likely that the increase in E in day-3 eggs is due, in part, to the breakdown of polar conjugates of E. Nine amino acids were present in significant quantities in eggs sampled at various times between 0 and 48 h post-oviposition. These include histidine, glutamine, proline, asparagine, serine, glutamic acid, threonine, lysine, and tyrosine. The first three amino acids were present at concentrations that were approximately 2 to 6 times greater than the concentrations of the last six amino acids. Amounts of most of the free amino acids varied with the age of the eggs from which the extract was prepared, but in general, there was no correlation between the levels at times of maximum parasitization (0 and 30 h) and the levels at the less favored times of parasitization (16 and 48 h). This information should facilitate the development of diets for both parasites and predators of pest species of beetles. Arch. Insect Biochem. Physiol. 44:172-182, 2000. Published 2000 Wiley-Liss, Inc.

Insecticidal Activity of Some Reducing Sugars Against the Sweet Potato Whitefly, Bemisia tabaci, Biotype B

Abstract 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 2nd 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.

In vitro rearing of Edovum puttleri, an egg parasitoid of the Colorado potato beetle, from egg to pupal stage in artificial diets devoid of insect sources: Effects of dietary amino acid and carbohydrate levels

Previous studies have demonstrated that Edovum puttleri, an egg parasitoid of the Colorado potato beetle, can be reared through the pupal stage in an artificial diet containing either hemolymph from Manduca sexta or CPB embryonic cell line-conditioned medium. In order to improve the diet and reduce production costs, i.e., eliminate the insect-derived components, the effects of 13 amino acids and 20 carbohydrates on the growth and development of the parasitoid were determined. In the presence of any one of five of the amino acids (arginine, glutamine, lysine, threonine and valine) at a concentration of 1%, or eight of the carbohydrates (cellobiose, fructose, gentiobiose, glucose, lactose, melinbiose, sorbitol and threhalose), also at a concentration of 1%, more than 75% of the larvae molted to the second instar. At higher concentrations (2 and 3%), all five of the amino acids and most of the eight sugars promoted third instar formation. Prepupal formation was also observed. In the presence of threonine (at 2 and 3%) or glutamine (at 3%), between 22 and 29% of the parasitoids formed prepupae. Of the six sugars (at 3%) that promoted prepupal formation, lactose and sorbitol which promoted 18 and 20% prepupation, respectively, were the most effective. In the presence of all 13 amino acids (each at 3%) or all 20 carbohydrates (each at 3%), 36 and 39%, respectively, of the parasitoids formed prepupae. At lower concentrations, reduced percentages of prepupal formation were observed. Since the amino acids, glutamine and threonine, and the sugars, fructose, gentiobiose, glucose, lactose, sorbitol and trehalose, when added individually to the diet (at a concentration of 3%) were most effective in promoting prepupal formation, the effect of one of these two amino acids in combination with each of the six sugars was tested. Glutamine in combination with lactose or sorbitol was able to promote pupation (≤6.3%), and threonine in combination with any one of five of the sugars (lactose and sorbitol were most effective) supported pupal formation (≤6.7%). Our study demonstrated that E. puttleri can be successfully reared from the egg through the pupal stage in an artificial diet devoid of insect materials.

Critical Feeding Periods for Last Instar Nymphal and Pharate Adults of the Whiteflies, Trialeurodes vaporariorum and Bemisia tabaci

Abstract 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°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 (≥ 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.

Irradiation of Colorado potato beetle eggs—effect on parasitism by the wasp Edovum puttleri

Summary In order to extend the period of time during which Colorado potato beetle (CPB) eggs are suitable hosts for the parasitic wasp Edovum puttleri, CPB eggs were subjected to various doses of gamma radiation between 2.5 x 104 and 5.0 x 106 rads prior to storage. For given storage times there were specific doses of radiation that resulted in notably, but not significantly, higher percent absolute emergence (number of adult wasps that emerged/total number of CPB eggs exposed to E. puttleri). After 7 days of storage, absolute emergence was highest after exposure to 2.5 x 104, 4.4 x 105 and 1.2 x 106 rads; and after 14 days of storage it was highest after exposure to 4.4 x 105 and 1.2 x 106 rads. For these doses of radiation and storage times, percent absolute emergence was equal to 40 ± 8%, a value that overlaps the range reported for CPB eggs that have not been irradiated. With increased storage time, absolute emergence dropped below 25%. While storage of irradiated eggs had a negative effect on percent parasitization, it did not affect the percentage of wasps that emerged (percent emergence = number of adult wasps that emerged/number of CPB eggs that were parasitized) from parasitized eggs. Percent emergence from non-irradiated eggs has been reported to average 78%, while in these studies, percent emergence from irradiated eggs typically was greater than 90%. Irradiation and storage of CPB eggs had little effect on the weights of adult wasps, and these wasps appeared to be active and in good condition. However, for unfed adult wasps, longevity decreased with increasing doses and storage times. Gamma irradiation of CPB eggs can be used to kill developing CPB embryos and to prevent deterioration of the host eggs. Our results show that irradiation can increase the period of time (from 72h to 14 days) in which CPB eggs are suitable hosts for rearing E. puttleri.