Orthogonal array design for optimization of an artificial medium for in vitro rearing of Trichogramma dendrolimi

Successful complete development of Trichogramma species on artificial media is also related to the presence and proportions of ingredients other than host insect‐derived components. In this study, an orthogonal array with six factors at three levels was performed and parameters of parasitism, larval development, pupation, and adult emergence were monitored to reveal the most important components of an artificial medium and to improve the medium for in vitro mass rearing of Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae). Results indicated that biological parameters of T. dendrolimi were affected differently by six ingredients of the artificial medium: pupal hemolymph of Antheraea pernyi (Guérin‐Méneville) (Lepidoptera: Saturniidae), egg yolk, 10% malted milk solution, Neisenheimer's salt solution, trehalose, and sterile water. Statistical analysis indicated that trehalose and Neisenheimer's salt solution, 10% malted milk solution, and pupal hemolymph of A. pernyi were the main ingredients of the artificial medium based on rates of parasitism and pupation, the number of larvae developing in each artificial egg, adult emergence rate, and the number of normal adults produced. A follow‐up bioassay with a selection of optimized formulas confirmed the validity of the optimization as predicted by the orthogonal array analysis, indicating the usefulness of this method for selecting artificial diets for entomophagous insects. Adult emergence rate of the parasitoid and total number of normal adults produced per egg card (each containing 20 artificial eggs) averaged 88.8% and 956 females on the best performing optimized artificial medium, consisting of 3 ml pupal hemolymph of A. pernyi, 2.5 ml egg yolk, 1 ml 10% malted milk solution, 1 ml Neisenheimer's salt solution, 0.1 g trehalose, and 1.5 ml sterile water. The latter medium was superior to any formerly developed medium and may thus have potential for the in vitro mass rearing of T. dendrolimi.     

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 parasitoid's 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.     

Antifungals: Mold control and safe levels in artificial media forTrichogramma [Hymenoptera: Trichogrammatidae]

Ten chemicals known to be fungicides are tested in artificial media forTrichogramma dendrolimi Matsu. [Hymenoptera: Trichogrammatidae]. Tests are performed in France by using artificial host egg capsules. Most of the fungicides cannot control fungus or are toxic forTrichogramma at effective levels. Geneticin, Amphotericin B and Nystatin, used at proper levels are able to control molds efficiently without modifyingTrichogramma development. These antifungals are also tested in actual conditions of mass production, in Guangzhou (China).      

Suitability assessment of three Trichogramma species in the control of Mythimna separata (Lepidoptera: Noctuidae)

Mythimna separata (Walker) is a serious cosmopolitan pest. Trichogramma species have been used worldwide as biological control agents of lepidopteran pests. However, no data on the control efficiency of Trichogramma species on this pest have been reported. We evaluated the suitability of M. separata eggs for three Trichogramma species, Trichogramma dendrolimi Matsumura, T. ostriniae Pang et Chen and T. japonicum Ashmead, and compared the suitability with that of Corcyra cephalonica (Stainton) eggs. The parasitism rate, development time, emergence rate, female proportion, body size and egg load of female offspring indicated that the M. separata egg is a suitable host for the three Trichogramma species. On the other hand, the non‐emergence rate and the relationship between egg load and body size reflected that M. separata eggs are less suitable for the three Trichogramma species than C. cephalonica eggs. Egg resorption occurred in T. japonicum 4 days after emergence.     

Effect of fertilized,unfertilized, and UV‐irradiated hosts on parasitism and suitability for Trichogramma parasitoids

Trichogramma spp. (Hymenoptera: Trichogrammatidae) parasitoids have been commonly used as biological control agents in insect pest management. Host quality is believed to influence parasitism, host preference, and suitability for parasitoids. To date, limited studies have compared the parasitism of Trichogramma parasitoids on fertilized, unfertilized, and sterilized host eggs. Hence, we studied the performance of three Trichogramma egg parasitoids, Trichogramma japonicum Ashmead, Trichogramma chilonis Ishii, and Trichogramma leucaniae Pang & Chen, on fertilized, unfertilized, and ultraviolet (UV)‐irradiated fertilized (UVF) eggs of rice moth, Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae). In a no‐choice test, T. japonicum and T. leucaniae parasitized significantly more fertilized or UVF than unfertilized hosts, and T. chilonis parasitized significantly more UVF than either fertilized or unfertilized hosts. In a choice test, all three Trichogramma parasitoids parasitized UVF hosts the most, and unfertilized hosts the least. There were similar percentages of adult emergence and female progeny among fertilized, unfertilized, and UVF hosts for all three Trichogramma parasitoids, except that T. japonicum had significantly lower adult emergence on fertilized hosts. We also found that all three Trichogramma parasitoids developed slower on unfertilized hosts. Regardless of host treatments, T. leucaniae had the longest developmental time and T. chilonis had the shortest. We conclude that Trichogramma parasitoids prefer parasitizing UVF eggs of C. cephalonica without negative effects on their emergence and sex allocation.     

Carbohydrate metabolism during the pupal molt of the tobacco hornworm,Manduca sexta

During the pupal molt of the tobacco hornworm, Manduca sexta, the percentage of active fat body glycogen phosphorylase increased from 5–10 to 20%, but only for a period of 5 h prior to the molt. From the time of the appearance of two sclerotized dorsal bars to the time of the molt, the concentration of total hemolymph carbohydrates doubled to 100 mM trehalose. Initially, the glucose level was high (16 mM) when compared with feeding larvae (approximately 1 mM) but decreased to zero just prior to the molt. The amount of cuticular chitosan decreased from approximately 100 mg to 10 mg at pupation; the exuvia contained approximately 7 mg. While the levels of total lipids in hemolymph were not affected, the lipid content of the fat body decreased significantly prior to the molt but increased sharply thereafter. Fat body glycogen phosphorylase in pharate pupae and pupae of M. sexta was substantially activated by the Manduca adipokinetic peptide hormone, which in pharate pupae, produced the same response at 2 and 20 pmol per insect as in ligated larval abdomens. In pupae the response was clearly reduced. Using chilling to stimulate glycogen phosphorylase, it was found that the enzyme in pharate pupae and pupae responded both in vivo and in vitro as in ligated abdomens of larvae. Thus, a transition to the adult response seems to occur during the pupal and pharate adult development. © 1995 Wiley-Liss, Inc.     

In vitro rearing ofTrissolcus basalis [Hym., Scelionidae] an egg parasitoid ofNezara viridula [Hem., Pentatomidae]

In vitro rearing of the egg parasitoidTrissolcus basalis (WOLL.) from eggs collected on the natural hostNezara viridula (L.) was initiated. Several oligidic diets containing insect material (Manduca sexta hemolymph or host egg content) were tested. Our initial medium with 50% hemolymph induced a high egg mortality, but by decreasing the hemolymph concentration, increasing the hen egg yolk concentration and adding 15% of free amino acids mixture, a hatching rate of 85% of the parasitoid eggs was obtained with 39% reaching the second instar and 33% the third instar. In a medium without hemolymph, but with 18% liquid from parasitized host eggs we obtained 90% to 100% hatching, 25 to 27% reaching the second instar and 8% the third instar. We did not obtain pupation from eggsin vitro, but did get pupae and adults from larvae rearedin vivo to second instar and transfered to anin vitro system.      

Hemolymph of reproductives of Solenopsis invicta (Hymenoptera: Formicidae)-amino acids,proteins and sugars

Free amino acid composition and carbohydrate and protein concentration of the hemolymph of the pupal and adult stages of reproductives of Solenopsis invicta are presented. The physico-chemical properties of the hemolymph differ between both sexes of fire ants during development. Male alates (459 mmol/kg) have a higher osmolality than pupae (388 mmol/kg). Osmolality during the pupal stage was 428 mmol/kg, decreasing to 354 mmol/kg soon after de-alation and increasing again to 463 mmol/kg in active queens. The hemolymph of newly eclosed adults was more basic than the pupa and older adults. The pH of the hemolymph ranged from 6.40 to 6.71 for males and 6.47 to 7.01 for females. An increase in the carbohydrate and protein content was found for both sexes after emergence. Newly de-alated females had a decrease in their carbohydrate titers, although no changes were found in the protein concentration. An increase in the total free amino acid concentration was recorded for males after emergence, while changes in females were noted only after de-alation. However, females had a 2.7-fold increase in the free amino acid pool after they de-alated, with proline, glutamine and taurine showing the highest increase. Changes in the organic composition and in the physico-chemical properties of the hemolymph of S. invicta are discussed in regard to the physiological processes involved in the maturation of the reproductives of this insect.     

Tyrosine and catecholamine levels in the hemolymph of tobacco hornworm larvae,Manduca sexta,parasitized by the braconid wasp,Cotesia congregata,and in the developing parasitoids

Parasitism of fifth instar Manduca sexta larvae by the gregarious parasitoid Cotesia congregata prevented normal storage of tyrosine in the hemolymph, whereas total tyrosine levels increased over eight times in the hemolymph of unparasitized larvae by day 4. Tyrosine glucoside, the hemolymph storage form of tyrosine and the precursor for pupal cuticle sclerotizing agents, was found only in trace amounts in parasitized larvae at the time of parasitoid emergence, but had increased to over 6 mM in hemolymph of unparasitized larvae. Concentrations of dopamine and N-β-alanyldopamine (NBAD), precursors for melanization and sclerotization of cuticle, respectively, had approximately doubled in the hemolymph of parasitized larvae by the day of parasitoid emergence, but not in unparasitized larvae. Catecholamine biosynthesis may be transiently stimulated for wound-healing, as black melanic pigmentation appeared around the wasp emergence holes in the host integument. C. congregata larvae accumulate tyrosine, dopamine, and NBAD by the time of emergence and cocoon spinning, either by direct uptake or by synthesis from precursors obtained from the host. NBAD increased in parasitoid larvae close to pupation, suggesting it functions as the main precursor for pupal cuticle tanning. Both dopamine and NBAD increased dramatically in pharate adult wasps just before eclosion and N-acetyldopamine (NADA) appeared for the first time. Dopamine was highest in concentration and total amount, and it can serve both as a precursor for black melanic pigmentation of adult wasp cuticle and for synthesis of NADA and NBAD, the precursors for cuticle sclerotization. Arch. Insect Biochem. Physiol. 38:193–201, 1998. © 1998 Wiley-Liss, Inc.     

Are egg parasitoids of the genus Trichogramma (Hymenoptera: Trichogrammatidae) promising biological control agents for regulating the invasive Box tree pyralid,Cydalima perspectalis (Lepidoptera: Crambidae)?

The Box tree pyralid, Cydalima perspectalis Walker (Lepidoptera: Crambidae), is an invasive species in Europe, not yet regulated by natural enemies. It is highly destructive to ornamental and native Buxus due to defoliation by larval feeding. The ability of eight Trichogramma species (T. brassicae Bezdenko, T. bourarachae Pintureau & Babault, T. cacoeciae Marchal, T. cordubensis Vargas & Cabello, T. dendrolimi Matsumura, T. evanescens Westwood, T. nerudai Pintureau & Gerding and T. pintoi Voegele) to parasitise and locate eggs of C. perspectalis was assessed in the laboratory. In direct observation tests, all species started to examine and oviposit into exposed eggs. Resulting egg parasitism was low and ranged between 4% and 20% for most of the parasitoid species after three days of exposure. Only T. dendrolimi parasitised significantly more host eggs with parasitism rates above 40%. On average, two to three progeny emerged from one host egg and the resulting sex ratio was female-biased, indicating suitability of the target host for progeny production. In subsequent cage tests on potted Buxus plants, again T. dendrolimi was the most efficient species in locating host eggs on foliage. We conclude that C. perspectalis eggs are attractive to various Trichogramma species. T. pintoi had the highest number of egg encounters and active females. T. dendrolimi females were most able to locate host eggs and to oviposit and produce successfully on them. Nonetheless, further study is needed to clarify the suspected role of potentially egg defensive mechanisms that may impede higher parasitisation in C. perspectalis.     

Comparative analysis of proteome maps of silkworm hemolymph during different developmental stages

Background  

The silkworm Bombyx mori is a lepidopteran insect with four developmental stages: egg, larva (caterpillar), pupa, and adult. The hemolymph of the silkworm is in an open system that circulates among all organs, and functions in nutrient and hormone transport, injury, and immunity. To understand the intricate developmental mechanisms of metamorphosis, silkworm hemolymph from different developmental stages, including the 3^rd day of fifth instar, the 6^th day of fifth instar, the 3^rd day of pupation, the 8^th day of pupal stage and the first day of the moth stage, was investigated by two-dimensional electrophoresis and mass spectrometry.     

在受重金属Zn2+胁迫的斜纹夜蛾幼虫寄主上双斑侧沟茧蜂的生存与发育

通过在植食性昆虫斜纹夜蛾Spodoptera litura Fabricius幼虫人工饲料中添加不同浓度的Zn²⁺,测定了连续3代取食含不同Zn²⁺浓度饲料幼虫的血淋巴中Zn²⁺含量,并研究了被Zn²⁺胁迫的斜纹夜蛾幼虫对寄生性天敌双斑侧沟茧蜂Microplitis bicoloratus Chen生存与发育的影响。结果表明,斜纹夜蛾幼虫血淋巴中Zn²⁺含量随饲料中Zn²⁺浓度和幼虫取食世代数的增加而增加;Zn²⁺胁迫斜纹夜蛾幼虫对双斑侧沟茧蜂的寄生率、从寄生到结茧的时间、蛹期长短、成虫羽化率和成虫寿命等都有不同程度的影响,其中以第2代的寄生率和第1代的羽化率受影响最大,均随着Zn²⁺浓度的增大而降低。因此,寄主饲料中高浓度的Zn²⁺能通过食物链影响双斑侧沟茧蜂的生存和发育。     

Aerodynamic separation of parasitised from unparasitised insect host eggs

Aerodynamic sorting of host eggs exposed to Trichogramma egg parasitoids improved the percentage parasitism of retained product. Losses of parasitised eggs declined as percentage parasitism of the unsorted eggs increased. Trichogramma eclosion from parasitised eggs was similar for the unsorted and retained fractions; emergence from the parasitised culls was significantly lower.     

Biological characteristics of three Trichogramma species on the eggs of diamondback moth (Plutella xylostella L.)

In this study, parasitism rate, development, emergence rate, sex ratio and adult longevity of Trichogramma brassicae, Trichogramma pintoi and Trichogramma embryophagum were investigated on eggs of diamondback moth, Plutella xylostella (Lep. Plutellidae) under controlled conditions. At the beginning of the experiments, 50 eggs of P. xylostella were exposed to parasitism of female wasps of different Trichogramma species. The eggs were replaced daily for three days and exposed eggs were kept in discrete glass vials. According to the obtained results, parasitism rates of T. brassicae, T. pintoi and T. embryophagum were 79.10, 93.90 and 86.10%, respectively. The adult emergence rates showed significant differences among various parasitoid species and were 93.72, 88.16 and 89.93% for T. brassicae, T. pintoi and T. embryophagum, respectively. No significant variations were observed among sex ratio values of the three parasitoid species. Consequently, results of present study suggested that T. pintoi could be more efficient for biocontrol of P. xylostella.     

Sublethal effects of indoxacarb on the diamondback moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis> (L.) (Lepidoptera: Yponomeutidae)

The effects of sublethal concentrations of indoxacarb on Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) were examined. In comparison with the control, sublethal concentrations led to a significant decrease in adult emergence, the percentage of pupation and pupal weight of the parent generation, fecundity, and adult longevity. They also significantly decreased the net reproductive rate (R ₀), intrinsic rate of increase (r _m), finite rate of increase (λ), and gross reproduction rate (GRR), and had significantly increased egg developmental time and doubling time (Dt) of P. xylostella. However, sublethal concentrations of indoxacarb had no significant affect on adult emergence, percentage of pupation, larva, prepupa and pupa developmental periods, hatchability, and sex ratio of offspring. The results of this study indicated that sublethal concentrations can decrease the biological parameters of P. xylostella.     

The life histories of two species of Sialidae (Megaloptera) from Japan

The life histories of two species of Sialidae (Megaloptera) from Japan were investigated, focusing on their larval climbing and emergence conditions and then adult emergence behaviours. At the Rokuman pond, a fence trap and pitfall traps were constructed beside the pond to collect final-instar larvae of Sialis yamatoensis and Sialis japonica as they climbed for pupation in the night. In the two species, 48–49% of climbings occurred under wet ground conditions, due to rainfall during or before the climbing night. Approximately 50% of larval climbings occur under dry ground conditions, during continuous sunny or cloudy weather. This is thought to be due to pupation occurring close the edge of the pond. Male and female adult emergences were nearly simultaneous in both species. Pupal periods varied, later final-instar larvae climbings resulted in shorter pupal periods. Ground temperature is thought to have an effect on pupal periods.     

Effects of short-term heat stress on the growth and development of Bradysia cellarum and Bradysia impatiens

Bradysia cellarum Frey and Bradysia impatiens Johansen are major pests of vegetable crops, as well as edible mushrooms and ornamental plants, and damage to hosts resulting in economic losses. Temperatures above the optimum levels for these pests have been predicted to regulate their population growth during summer. The aim of the present study was to examine the effects of both heat stress and exposure time on the growth and development of eggs, larvae and pupae for two Bradysia species. The egg stage, egg hatching rate, 4th instar larval stage, pupation rate, pupal stage and adult emergence rate were observed after exposing at high temperatures of 34°C, 37°C and 40°C for 1, 2, 4 and 6 hr. The results showed that 34°C, 37°C and 40°C for 1-, 2-, 4- and 6-hr exposure treatments prolonged the developmental stage of egg, 4th instar larva and pupa, while decreasing the egg hatching rate, pupation rate and adult emergence rate. This suggests that increasing temperature or prolonging exposure time to the heat stress could significantly affect insect survival, growth and development. Our study could provide an ecological basis for pests’ management strategy by using short-term heat stress.     

Laboratory rearing of immature Culicoides peregrinus Kieffer,a potential vector of bluetongue virus

Culicoides peregrinus (Kieffer) (Diptera: Ceratopogonidae) was reared from egg to adult using four different combinations of food and substrate (T1: absorbent cotton, mud broth with 2% yeast; T2: 1% agar, mud broth with 2% yeast; T3: absorbent cotton, nutrient broth; T4: absorbent cotton, 2% yeast). Field‐collected engorged females exhibited mean fecundity of 82.45 ± 4.00. The highest rate of emergence and largest adults were obtained in T1, and the lowest rate of emergence was observed in T4. Two‐way analyses of variance with post hoc Tukey tests showed significant differences in age at pupation, pupal weight and wing length among the various treatments, although the sex ratio was 1 : 1 in all food/substrate combinations. The successful rearing of immature C. peregrinus is an encouraging step towards the establishment of a laboratory colony of this prevalent species associated with livestock.     

In VitroRearing ofTrichogramma minutumRiley (Hymenoptera: Trichogrammatidae) for Ten Generations, with Quality Assessment Comparisons ofin Vitroandin VivoReared Adults

Trichogramma minutumRiley were reared for 10 generations on an artificial diet containing a yeast extract, FreeAmine III, nonfat dry milk, chicken egg yolk, chicken embryo extract, andManduca sexta(L.) egg liquid. Quality control parameters, including adult longevity, sex ratio, pupation rate, percentage of pupae to emerge as adults, adult female body length, number ofHelicoverpa zea(Boddie) eggs parasitized by a female, and percentage of deformed females were assessed and compared to insects rearedin vivoon irradiatedH. zeaeggs. The development time was longer forin vitroreared insects, but there were more deformed females in thein vitroculture. The sex ratio, however, was generally not significantly different between thein vitroandin vivocultures. Thein vitroreared females generally were larger, lived longer, and parasitized moreH. zeaeggs. Emergence of adults was in excess of 75% in all but the firstin vitrogeneration and was generally not significantly different from adult emergence in thein vivoculture. These findings will be of value in the development of a practical system forin vitromass rearing ofTrichogrammafor use in biological control.     

Refinement of a cell line based artificial diet for rearing the parasitoid wasp, Trichogramma pretiosum

An artificial diet incorporating insect cells originally developed for Trichogramma australicum Girault (Hymenoptera: Trichogrammatidae) was successfully used to rear Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae). To refine the diet, individual components were removed. Chicken egg yolk and the insect cells were identified as the most important components for T. pretiosum development. Their removal resulted in few pupae and no adults. Removal of Grace’s insect medium, a common component of artificial diets, was found to markedly improve the development of T. pretiosum, producing 60% larva to pupa transition and 19% pupa to adult transition. There was no significant difference in T. pretiosum development on diets in which milk powder, malt powder or infant formula were interchanged, despite differences in nutrient composition. The use of yeast extract resulted in significantly higher survival to the adult stage when compared with yeast hydrolysate enzymatic and a combination of yeast extract and yeast hydrolysate enzymatic. Comparison of four antimicrobial agents showed the antibacterial agent Gentamycin and the antifungal agent Nystatin had the least detrimental effect on T. pretiosum development. The use of insect cell line diets has the potential to simplify artificial diet production and significantly reduce T. pretiosum production costs in Australia compared to diets using insect hemolymph or the use of natural or factitious hosts.