Interaction of calyx fluid and venom from Microplitis croceipes (Braconidae) on developmental disruption of the natural host, Heliocoverpa zea, and two atypical hosts, Galleria mellonella and Spodoptera exigua.

Polydnaviruses of many braconid and ichneumonid endoparasitoids play an important role in the successful parasitism of their hosts. The host's development is altered and its immune response is also suppressed. In this study, we compared the effects of calyx fluid and venom on the development of the natural host, Helicoverpa zea, and two atypical hosts that the parasitoid does not normally attack in nature, Galleria mellonella and Spodoptera exigua. The levels of calyx fluid and\or venom injected was 0.05, 0.1 and 0.2 female equivalents (FE)/larva. In H. zea, calyx fluid significantly reduced larval growth on day 5 post injection. Venom alone did not affect larval growth but it synergized the action of calyx fluid by reducing growth earlier and for a longer period after injection. Other effects of calyx fluid on the host, either alone or in combination with venom, were an increase in developmental period, and a reduction in percent emergence and weight of adult moths. The percentage of H. zea larvae that pupated was not affected by calyx fluid or venom. In Galleria mellonella, venom alone reduced larval growth comparable to calyx fluid and both tissues induced the effects on day 1 post injection. Other effects caused by calyx fluid or venom alone or the combination were a reduction in percent pupation and emergence, and the average adult weight. In S. exigua, high mortality occurred when 4th instar larvae were injected. Although the injection of larger fifth instars reduced overall mortality, the sham-injected larvae only gained weight during the first 24 hours after injection (from day 0 to day 1). However, adults were produced at all doses of calyx fluid or venom. The effects of the virus on development in this species were a prolongation of the larval stage and reduction of adult weight by calyx fluid in combination with venom. In conclusion, injections of calyx fluid and venom of Microplitis croceipes can differentially affect the growth and development of its natural host H. zea, and atypical host, G. mellonella, but only a minimal effect was observed in S. exigua.

Multicellular-vesicle-promoting polypeptide from Trichoplusia ni: tissue distribution and N-terminal sequence.

An N-terminal amino acid sequence of a 16.9 kDa hemolymph polypeptide, "Vesicle Promoting Factor" (VPF) from Trichoplusia ni, revealed a high sequence homology (70%) with Manduca sexta apolipophorin-III. A polyclonal antibody developed against VPF, however, was not immunoreactive with either purified M. sexta or T. ni apolipophorin-III. Immunoblots of tissue homogenates of T. ni indicated that VPF was present in imaginal wing discs, central nervous system (CNS), silk glands, midgut and hemocytes from fifth instar larvae, and also in the IAL-TND1 cell line which can grow as either fluid-filled multicellular vesicles or multicellular aggregates. VPF was also detected immunologically in the hemolymph of adults of T. ni, and in hemolymph of adults and larvae of Galleria mellonella and Heliothis virescens. Testes, midgut, hemocytes, and wing discs, but not Malpighian tubules, of T. ni released VPF into tissue culture medium during a 3 h incubation period.

Embryonic development of an endoparasitoid, Microplitis croceipes (Hymenoptera: Braconidae) in cell line-conditioned media.

Embryos of the parasitoid Microplitis croceipes develop from pregerm band stage to first larval instar in cell culture medium conditioned by a cell line (IPLB-LdFB) derived from fat body from an atypical host Lymantria dispar. However, the percentage of eggs that develop normally to the first larval instar stage is significantly less than for those maintained in IPL-52B medium conditioned with host fat body tissue. Therefore, we examined the capacity of five insect cell lines to promote growth and development of pregerm band eggs in five media, IPL-52B, TC-199, TC-100, Grace's, and ExCell 400. The developmental response of M. croceipes was dependent both on the cell line and the cell culture medium used. TC-100, TC-199, and Grace's media promoted development to the germ band stage without the need for conditioning with host tissue. IPL-52B supported development to the germ band stage when a defined lipid concentrate was added. In IPL-52B medium, the IPLB-LdFB cell line promoted a significantly higher number of eggs developing to germ band relative to the other cell lines; however, none of the cell line-conditioned IPL-52B medium significantly stimulated egg hatch relative to the control medium. None of the cell line-conditioned Grace's media had a significant effect on eggs attaining germ band stage compared with the Grace's control medium. However, Grace's medium conditioned with the IAL-TND1 and IPLB-LdFB cell lines promoted development beyond germ band, resulting in a significantly higher percentage of hatching eggs than the Grace's control medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth factors in invertebrate in vitro culture.

An increasing number of polypeptide growth factors have been identified that have proven essential in the development of defined cell culture media for mammalian cell culture. The development of defined mammalian cell culture media, in turn, has provided an environment for studying cell lines in an experimentally manageable unit for studying the action of cellular regulators and genes that determine the properties of cells. Evidence that vertebrate growth factors may be present in insects is based on DNA sequences that encode epidermal growth factor and transforming growth factor-beta. However, research on the influence of commercially available vertebrate growth factors is very limited. Although the majority of insect growth-promoting substances studied were isolated directly from insect hemolymph, few of these have been purified to the extent that they could be tested in insect cell, tissue, and endoparasite cultures. Research is needed in both of these areas to aid in developing defined insect culture systems, and to understand better the regulation of postembryonic growth and development in insects.

Stimulation of embryonic development in Microplitis croceipes (Braconidae) in cell culture media preconditioned with a fat body cell line derived from a nonpermissive host, gypsy moth, Lymantria dispar.

A cell culture medium, IPL-52B, was preconditioned with host fat body and two insect cell lines to determine if they would support embryonic development of Microplitis croceipes in vitro. The medium was preconditioned with the cell line IPL-LdFB, derived from fat body of the gypsy moth, Lymantria dispar, cell line IAL-TND1, derived from imaginal discs of the cabbage looper, Trichoplusia ni, and whole fat body tissue from host Helicoverpa zea. A second cell culture medium, Excell 400, was preconditioned with only the cell line, IPL-LdFB. Pregerm band eggs were dissected from third instar host larvae and incubated in the conditioned medium for 20 h. Newly laid parasitoid eggs did not develop in unconditioned IPL-52B, but did develop to germ band stage in unconditioned Excell 400. The IPL-52B medium conditioned with both cell lines induced germ band formation, but only the L. dispar cell line (IPL-LdFB) promoted significant development to eclosion comparable to host far body tissue. Excell 400 medium preconditioned with the cell line, IPL-LdFB also supported development to eclosion.

Effects of female wasp accessory secretions, host fat body, and host hemolymph on protein synthesis and egg viability in Microplitis croceipes (Braconidae).

Effects of female wasp reproductive gland secretions, host fat body and hemolymph, and mechanical constriction of the parasitoid egg on protein synthesis were studied in eggs of Microplitis croceipes (Braconidae) dissected from the wasp ovary. Protein synthesis was measured by 35S-methionine incorporation in eggs held in tissue culture medium for 16 h after treatment. Synthesis was stimulated in oocytes obtained from three regions of the ovary (egg tube, reservoir, and calyx) by fat body and venom gland but not by calyx fluid. A combination of fat body, venom gland, and calyx fluid did not enhance the level of synthesis relative to that of fat body or venom gland alone. Host hemolymph inhibited protein synthesis when incubated directly with the dissected eggs but not when the eggs were collected from an artificial oviposition substrate (AOS) containing hemolymph. The inhibitory effect of the hemolymph is thought to be due to the occurrence of melanization. Mechanical constriction did not alter the rate of synthesis, confirming an earlier report that synthesis in newly deposited eggs in ongoing and is not dependent on mechanical activation during the act of oviposition. Mechanisms responsible for sustaining protein synthesis in eggs for 16 h in vitro after their exposure to host hemolymph in the AOSs or fat body and venom gland are not known. Only a small percentage (less than 2%) of dissected ovarial reservoir oocytes that were mechanically constricted and exposed to the venom gland, calyx fluid, and host fat body hatched in vitro. In contrast, an earlier study demonstrated that 38% of eggs oviposited by female wasps into AOSs developed and hatched.

Pheromone hydrolysis by cuticular and interior esterases of the antennae, legs, and wings of the cabbage looper moth,Trichoplusia ni (Hübner).

Examination was made of the hydrolytic activities of esterases obtained from the antennae, legs, and wings of 3-day-old cabbage looper moths,Trichoplusia ni (Hübner), by elution and by homogenation of those appendages. Pheromone hydrolysis in 1-min assays was monitored by use of tritium-labeled (Z)-7-dodecen-1-ol acetate and thin-layer chromatography to separate the reaction products. Listed according to the activities of the esterases obtained by homogenation, the organs were antennae > legs > wings. In contrast, the order according to the activities of the eluted esterases was wings > legs > antennae. Also, the eluted enzymes were less active than the esterases obtained by homogenization. The relatively high pheromone-hydrolyzing activity present in homogenized antennae suggests that the esterases originated inside the antennae and lends support to the hypothesis proposed in earlier investigations that pheromone-inactivating enzymes may play an important role in the olfactory process, possibly by clearing pheromone from the vicinity of the olfactory receptors. The esterases detected on the cuticle, on the other hand, may function by preventing surface accumulation of pheromone. The higher measured esterase activity in homogenates of prothoracic legs than of mesothoracic or metathoracic legs suggests that the prothoracic legs, which are used to clean the antennae of debris, may function by removing and degrading pheromone from the surface of antennae.

Chitin biosynthesis in imaginal discs cultured in vitro.

We have investigated the stimulation of cuticle production by imaginal discs ofPlodia interpunctella in tissue culture. We turned to biochemical methods to assess the quantitative effects of beta-ecdysone on chitin biosynthesis in wing discs incubated with 0.5 µC of C14-glucosamine for the final 24 h of culture.We demonstrated that imaginal discs ofP. interpunctella respond to increasing concentrations of ß-ecdysone with increased synthesis. The threshold is between 0.01 and 0.1 µg/ml of hormone (2×10-8 M to 2×10-7 M). These data represent the first demonstration of quantitative biosynthesis of chitin by a developing tissue in vitro in relation to varying amounts of hormone. Additionally, protein synthesis during the ß-ecdysone-dependent period was necessary for chitin synthesis. This system thus lends itself to a detailed investigation of the control of chitin biosynthesis.

Influence of a haemolymph protein fraction on the binding of juvenile hormone in homogenates of insect epidermis (Plodia interpunctella (Hübner)).

A carrier protein fraction (CPF) from larval haemolymph was found to influence binding and catabolism of tritiated juvenile hormone (JH) in homogenates of larval epidermis. The CPF reduced binding of tritiated JH in all of the particulate fractions but did not alter the relative binding pattern when compared with JH alone. The CPF also protected the hormone from degradative enzymes in the membrane vesicle and microsomal + cytosol fractions but not in the nuclear and mitochondrial fractions. Preliminary evidence exists for high-affinity binding sites for JH in the nuclear and mitochondrial fractions. We conclude that the CPF influences catabolism of the tritiated JH but does not participate in subcellular recognition of JH in homogenized target tissue.

Energy transduction: inhibition of cockroach feeding by naphthoquinone.

1,4-Naphthoquinones inhibit feeding of Periplaneta americana by complexing with sulfhydryl groups of receptor protein in sensory neurons, by oxidizing the sulfhydryl groups, and by being reduced.