Stephen M. Ferkovich

Growth factors in invertebrate in vitro culture

SummaryAn 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-β. 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.

A study of uptake of radiolabeled host proteins and protein synthesis during development of eggs of the endoparasitoid, Microplitis croceipes (Cresson) (Braconidae)

Abstract The uptake of radiolabeled haemolymph and fat body proteins from fourth instar larvae of Heliothis zea (Boddie) by eggs of Microplitis croceipes (Cresson) was examined by SDS-polyacrylamide gel electrophoresis and by autoradiography. None of the 125 I-labeled haemolymph proteins was detected in eggs exposed to the proteins in vivo . Although several of the proteins were observed in eggs incubated with the labeled proteins in vitro , none of these proteins was degraded or resynthesized into new structural proteins during development of the embryo. Similarly, no significant uptake of labeled fat body proteins by the eggs could be detected in vitro . On the other hand, protein synthesis measured by incorporation of [ 35 S]methionine occurred throughout egg development. Proteins were synthesized at least 1 hr after the egg was deposited into the host. The protein patterns of eggs on one-dimensional SDS gels were complex and ranged in size from less than 18,500 to more than 330,000 mol. wt. The protein band patterns of the newly synthesized proteins showed some qualitative differences at 1–8, 16–32 and 40 hr after egg deposition. We conclude that eggs do not absorb or utilize the host apoproteins (or degradation products) but instead synthesize proteins de novo from free amino acids in the host haemolymph.

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 hosts 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 andor 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.

Chitin synthesis in larval and pupal epidermis of the Indian meal moth, Plodia interpunctella (Hübner), and the greater wax moth, Galleria mellonella (L.)☆

The level of chitin synthesis was determined in whole last-instar larvae and in pupae of Plodia interpunctella, and in epidermal tissue from similar stages of Galleria mellonella. The incorporation of radiolabelled N-acetyl-d-glucosamine into chitin was used to measure synthesis. Chitin production was similar in both species with peaks of synthesis occurring at the beginning of the last larval instar, in prepupae, in white pupae and prior to adult emergence. P. interpunctella also exhibited an additional small increase at mid-instar. Exposure of larval epidermis of P. interpunctella to 20-hydroxyecdysone in vitro stimulated chitin synthesis, but only after a 24 hr lag period subsequent to exposure to the hormone. This hormonal stimulation of chitin synthesis was inhibited by actinomycin-D and cycloheximide which suggested that 20-hydroxyecdysone-stimulated production of chitin depended on synthesis of RNA and protein. Comparison of the synthesis of chitin in epidermis of G. mellonella with previously published ecdysone titres, indicated that chitin production in vivo is preceded by an elevated ecdysone titre.

Sex pheromone of the cabbage looper: Reactions with antennal proteins in vitro☆

Abstract In the presence of (Z)-7-dodecen-1-ol acetate, the sex attractant of the cabbage looper, Trichoplusia ni, soluble protein from male antennae showed a time-dependent difference spectral absorbance at 280 nm. The change was associated with the enzymatic conversion of the pheromone to (Z)-7-dodecen-1-ol, a potent inhibitor of behavioural responses to the pheromone. In contrast, the response obtained with the inhibitor was indicative of non-enzymatic binding to specific protein(s) in the fraction. GLC analyses of the relative rates of enzymatic hydrolysis of the pheromone by the antennae, haemolymph, and legs revealed 33·9, 10·1, and 6·5 per cent conversion per hour, respectively. These results may provide an insight into the fate of a pheromone in the olfactory process of this insect; however, the significance of the reaction with the inhibitor is not yet known.

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

SummaryEmbryos of the parasitoidMicropolitis 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 hostLymantria 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 ofM. 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 hand 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. Although the BCIRL-HZ-AMI cell line, which is derived from the parasitoid’s typical host, did not induce hatch in either IPL-52B medium or Grace’s medium, it promoted hatch in TC-199 and Excell 400 media. Fat body taken from the same species that the cell lines were derived from was a better predictor of a cell line’s embryotrophic activity in Grace’s medium rather than in IPL-52B medium. Thus, the composition of the medium and the species and tissue type of the cell line source must be evaluated interactively to determine optimal conditions for promoting development ofM. croceipes in vitro.

Release of a juvenile hormone binding protein by fat body of the Indian meal moth, Plodia interpunctella, in vitro.

Abstract When fat body of fifth instar larvae of Plodia interpunctella was cultured in vitro in a chemically defined medium, the tissue released a low mol. wt protein (FBBP) that binds juvenile hormone (JH). This FBBP has the same mol. wt, estimated by gel permeation chromatography, as the haemolymph JH binding protein. Furthermore, the FBBP protected JH from degradation by general esterases isolated from the haemolymph. Treatment of fat body with cycloheximide inhibited incorporation of [ 14 C] leucine into the FBBP protein fraction and reduced the amount of FBBP released into the medium. We conclude that one source of the JH binding protein found in the haemolymph is the fat body.

Enhanced oviposition in the insidious flower bug, orius insidiosus (Hemiptera:Anthocoridae) with a partially purified nutritional factor from prey eggs

Abstract The insidious flower bug, Orius insidiosus (Say), can be maintained on a minimal artificial diet composed of brewers yeast, soy protein hydrolysate and chicken yolk. However, egg production is poor even though the level of protein in the diet exceeds the amount consumed by adults that are fed insect eggs and have higher levels of egg production. We therefore fractionated eggs of the almond moth, Ephestia kuehniella Zeller by preparative isoelectric focusing and bioassayed the resultant fractions in test diets. Ovipositional rates were evaluated using a short 1-week bioassay. Adult predators were placed on the diets the third day after eclosion, allowed to feed for six days, and then provided with an oviposition substrate for 24 h on day seven. Egg production significantly increased only in a fraction with an isoelectric point of pH 5. SDS-PAGE revealed the presence of several Commassie blue-stained bands; however, the nature of the factor is unknown. These results point to a fecundity factor required by females of O. insidiosus for egg laying that potentially may be used to supplement artificial diets for Orius species by commercial producers of beneficial insects.

Regulation of protein synthesis during egg development of the parasitic wasp, Microplitis croceipes (Cresson) (Braconidae)

Regulation of protein synthesis was studied in Microplitis croceipes (Cresson) during oogenesis and after oviposition in its host, Heliothis zea (Boddie). Oocytes were dissected from ovaries of M croceipes during various stages of development and from second instar host larvae at various times after egg oviposition. Uridine incorporation into RNA was highest in oocytes in the ovarian egg tube, then declined in oocytes present in the egg reservoir and calyx of the ovary. During blastoderm formation, 4–6 hr after the egg was oviposited into the host, uridine was still incorporated at low levels. At 18–24 hr after oviposition (14–18 hr after blastoderm formation), uridine incorporate returned to the level observed in eggs in the ovarian reservoir. Protein synthesis, measured by [3H]leucine and [35S]methionine incorporation was continuous at all stages of development and was most rapid 14–20 hr after blastoderm formation when RNA synthesis was highest. Patterns of polypeptide synthesis as analyzed by one-dimensional SDS-PAGE changed quantitatively but this method did not reveal any new polypeptides in oocytes in various stages of development or during early and late embryogenesis. However, when polypeptide patterns of eggs were compared on two-dimensional gels during embryogenesis, synthesis of new polypeptides was evident after cellularization of the blastoderm (4–6 hr after oviposition). These changes in synthesis after blastoderm formation may be the result of transcription and subsequent translation of embryonic mRNA.

Parasitism of a factitious host, Galleria mellonella (Lepidoptera:Pyralidae) by an endoparasitoid:oviposition and emergence of Microplitis croceipes (Hymenoptera:Braconidae)

The effect of various diet supplements on the development of Microplitis croceipes in an atypical host, Galleria mellonella (Linnaeus), were evaluated, as were ovipositional responses to various factors. Female parasitoids were exposed to fifth instar G. mellonella in Petri dishes containing the following treatments either separately or in combination: a) Helicoverpa zea (Boddie) frass, b) 1% and 10% solutions of a host-seeking stimulant (13-methylhentriacontane), c) H. zea hemolymph, and d) H. zea hemolymph concentrated by freeze-drying. There were no significant differences between hemolymph and frass + hemolymph treatments. The host-seeking stimulant alone also stimulated oviposition. The most effective combination was host-seeking stimulant and concentrated hemolymph which induced oviposition rates comparable to that in the typical host, H. zea. Various diet supplements did not improve the development and emergence of M. croceipes. We conclude that oviposition by M. croceipes in the atypical host, G. mellonella, was significantly improved by the application of host-seeking stimulant and concentrated hemolymph, but the rate of adult parasitoid emergence was not increased by the addition of nutrient supplements to the host diet.