Testicular and androgen dependence of skin gland morphology in the anurans,Xenopus laevis and Rana pipiens

In anuran amphibians, there is increasing evidence that exocrine glands dispersed throughout the general integument are secondary sex characters (SSC). Following the recent discovery of sexually dimorphic “breeding glands” in the dorsum of male Rana pipiens, we studied the effects of castration and testosterone treatment on the dorsal skin glands of male Xenopus laevis and R. pipiens to determine whether the dorsal breeding glands, or any other dorsal skin glands, are androgen dependent. The dorsal skin glands of X. laevis were unaffected by androgen status. By contrast, in R. pipiens, breeding, mucous, and seromucous glands responded to testosterone stimulation. Mucous glands were significantly (P < 0.05) larger in testosterone-treated frogs than in castrates. There was a large, but statistically insignificant, increase in the size of the dorsal breeding glands. Testosterone treatment also increased the epithelial cell height of breeding and seromucous glands (P < 0.05). In the skins of castrated and testosterone-treated frogs, there was a reciprocal relationship between the abundance of seromucous and breeding glands: in castrates, seromucous glands were abundant and breeding glands virtually absent, whereas in testosterone-treated frogs, breeding glands were abundant and seromucous glands less common. The total number of the two gland types was similar in both treatment groups. Glands that appeared to be intermediate in form between seromucous and breeding glands were observed in some frogs. These data suggest that seromucous glands may be the regressed form of breeding glands in the dorsal skin of R. pipiens and that the dorsal skin of R. pipiens is a SSC. © 1993 Wiley-Liss, Inc.     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 1. Comparison to the rat liver enzyme

The characteristics of the enzyme γ-glutamyltranspeptidase were determined in frog liver and compared to those of the rat. In Rana pipiens, tissue distribution studies indicated the order of activity to be: kidney >>> liver >> nerve > egg > lung > heart > skeletal muscle in homogenates. In the Rana pipiens relative to the Fischer 344 rat, the activity of the liver enzyme was somewhat greater (1·8-fold) and the kidney enzyme substantially less (25-fold). Frog liver γ-glutamyltranspeptidase displayed strain-dependent differences in activity with Rana pipiens and Rana sylvatica exhibiting comparable activities and Xenopus laevis exhibiting 20-fold lower activities. No influence of sex was apparent in Rana pipiens in contrast to the sex dependent differences observed in the Fischer 344 rat: ♀ : ♂ = 7:1. In homogenates and plasma membrane fractions of Rana pipiens, Xenopus laevis and the Fischer 344 rat, high, and comparable relative specific activities, were observed, 8–11, coupled with protein yields of 2·2–2·5 per cent indicating the enzyme to be plasma membrane bound and associated with the sinusoidal surface of the liver cell. Both the frog Rana pipiens and Xenopus laevis and Fischer 344 rat liver plasma membrane enzymes displayed comparable temperature-induced activation (1·51–1·74-fold) but with a peak for the frogs at 60°C and for the rat at 50°C. Both Acivicin and maleate inhibited the liver plasma membrane γ-glutamyltranspeptidase of both Rana pipiens and the Fischer 344 rat, but the frog enzyme was less sensitive (89 per cent decrease versus 97 per cent decrease) to 150 μM Acivicin and more sensitive (65 per cent decrease versus 35 per cent decrease at 150 mM maleate) to maleate. Kinetic studies indicated that the liver plasma membrane enzyme from Rana pipiens had a K_m of 0·61 mM and V_max of 55·6 nmol mg^?1 min^?1 and that from the Fischer 344 rat had a K_m of 3·57 mM and V_max of 71·4 nmol mg^?1 min^?1.     

Local alteration of cortical actin in Xenopus eggs by the fertilizing sperm

Rhodamine phalloidin (Rph) staining was used to examine the microfilament organization of the Xenopus laevis egg cortex during the early stages of fertilization. Unactivated eggs possessed a cytochalasin B (CR)-insensitive Rph-stained matrix that was reorganized upon egg activation and diminished in the presence of CB. Xenopus laevis sperm caused a temporary local increase in Rph staining on the Xenopus cortex. In CB-treated eggs, the local increases of cortical Rph staining later changed to a Rph-free area. These temporary local increases of cortical Rph staining were also observed when Notophthalmus viridescens sperm fertilized Xenopus and Rana pipiens eggs, and were followed by the appearance of concentric rings of stained and unstained areas. Our data suggest that Xenopus and Notophthalmus sperm have activities that can both organize and disrupt the cortical filamentous actin of the Xenopus egg. © 1993 Wiley-Liss, Inc.     

Ultrastructural changes associated with UV irradiation in the "germinal plasm" of Xenopus laevis

To detect structural changes following UV irradiation in the “germinal plasm,” ultrastructure of the “germinal plasm” was studied in normal and UV-irradiated eggs of Xenopus laevis at the following stages: prior to fertilization, early 2-cell, 32-cell, and late blastula. It was revealed that ultrastructural features of the “germinal plasm” were essentially common between Xenopus laevis and Rana pipiens. That is, the “germinal plasm” is composed primarily of a large aggregation of mitochondria and distinctive electron dense bodies (germinal granules). Irregularly shaped cylinderlike granules (giant germinal granules), having the same internal characteristics as the germinal granules, were found in the “germinal plasm” of all eggs examined.Comparison between normal and UV-irradiated eggs has demonstrated that UV irradiation causes swelling and vacuolation of mitochondria and fragmentation of germinal granules. The suggestion is that the integrity of certain UV-sensitive factor(s), which is involved in maintaining normal structure of germinal granules, is indispensable for the determination of the primordial germ cells.     

Circular mitochondrial DNA from Xenopus laevis and Rana pipiens

Mitochondrial DNA from oocytes of Xenopus laevis and Rana pipiens and from the liver of Gallus domesticus was studied by electron microscopy using the Kleinschmidt technique. A high percentage of circular molecules, either highly twisted or open, was observed in all preparations. The mean contour length of circles from X. laevis was 5.40 , from R. pipiens 5.56 and from G. domesticus 5.26 . Highly twisted circles were found in greater abundance in a fresh preparation than in preparations left standing for 3 months. These molecules are considered to be the native form of mitochondrial DNA.     

The satellite cells of normal anuran skeletal muscle

Population counts and size measurements of satellite cell nuclei and myonuclei were carried out on the normal gastrocnemius muscles of adult Rana pipiens and Rana clamitans. Satellite cell profiles occurred with an observed frequency of about 1.3% in the muscles of the R. pipiens, and with an observed frequency of about 1.6% in the muscles of the R. clamitans. These frequencies were not found to be significantly different. The observed frequencies were corrected for the sampling bias introduced by the difference in the mean size of the satellite cell nuclei and myonuclei. This correction suggests that R. pipiens and R. clamitans both have a true satellite cell frequency of approx 2.7%. Analysis of these data indicates that the satellite cells of the normal anuran gastrocnemius occur in sufficient numbers to account for the regeneration seen after injury to this muscle.     

The influence of diffusion in the external medium upon the determination of cytoplasmic diffusion coefficients

The exchange of isotopic water has been studied in ovarian eggs from three Anuran species (R. esculenta, R. pipiens and R. temporaria) by the automatic diver balance technique. The oocytes were treated with chemicals (digitonin, ethanol and formaldehyde) to remove the plasma membrane. The cytoplasmic diffusion coefficient for water was calculated with and without allowance for the influence of diffusion in the external medium. The former expedient was found necessary in order to obtain correct values. The chemical treatments gave different values, but there are sound reasons to believe that those obtained with digitonin (8.9, 15.3 and 10.8.10^?6 cm²sec^?1 for R. esculenta, R. pipiens and R. temporaria, respectively) are the correct ones. These results indicate that the rate of diffusion in the oocyte cytoplasm amounts to 50–75% of that of free diffusion.     

Pomphorhynchus laevis (Müller) in the flounder, Platichthys flesus L., in the tidal River Thames: population structure, microhabitat utilization and reproductive status in the field and under conditions of controlled salinity

The infection of R. Thames flounders, Platichthys flesus L., at Fulham by the acanthocephalan parasite Pomphorhynchus laevix (Müller) is described in terms of parasite population structure, life-cycle organization, reproductive biology and host microhabitat utilization. The parasites demonstrated 100% prevalence in this tidal but essentially freshwater locality (intestinal intensity 34.47) and were overdispersed in the flounder population (variance/mean ratio = 10.39, k= 1.04). Overall about 11% of the worms occurred in peritoneal cavity sites, the remainder being firmly attached to the gut wall in the posterior region of the intestine and rectum. The P. laevis populations were reproductively active, demonstrating that the flounder acts as a significant final host in this locality. Viable larval stages were identified in Gammarus zaddachi Sexton, a predominantly estuarine amphipod, indicating that P. laevis is likely to be able to complete its life cycle at Fulham. Over half the female parasites examined were gravid and many of the non-gravid worms were inseminated but had not yet started egg production. The proportion of females gravid, the number of ovarian balls and the number of eggs in gravid females was seen to increase with worm size. Both the identity of the intermediate host and the reproductive status of P. laevis in the Thames flounders differ from the Pomphorhynchus/flounder system studied by Kennedy (1984) in the R. Avon, suggesting that the two P. laevis populations may belong at least to discrete subspecies or strains. Parasites in flounders maintained under laboratory conditions in fresh water, 50% sea water and 100% sea water showed similar population, microhabitat and reproductive characteristics to those observed in the field. This suggests that increased salinity has a negligible effect on established parasites in the short term, and therefore that salinity may not form a barrier to the survival and dispersal of the R. Thames parasite when the flounders return to sea.     

Properties of a cytostatic factor from Xenopus laevis eggs.

The cytoplasm of mature eggs of Xenopus laevis was found to contain a cytostatic factor (CSF) which induces cleavage arrest at metaphase when microinjected into one blastomere of a two-cell embryo of Xenopus laevis or Rana pipiens. The Rana CSF was found to be incapable of arresting mitosis in Xenopus embryos. Both Xenopus and Rana CSF were stabilized during the transfer procedure by Ca²⁺-chelation in the donor egg. The Xenopus CSF was not present in the germinal vesicle of immature oocytes, but arose in the cytoplasm at the time of germinal vesicle breakdown and subsequently disappeared at the time of fertilization or egg activation.     

Protection of cowpea,Vigna unguiculata L. (Walp.) with Newbouldia laevis (Seem.) extracts against infestation by Callosobruchus maculatus (Fabricius)

Evaluation of protectant ability of Newbouldia laevis (Seem.) extracts against infestation by Callosobruchus maculatus in cowpea, Vigna unguiculata L. (Walp.) was carried out in the laboratory at ambient temperature of 28?±?2?°C and 70?±?5% relative humidity. Extracts from wood ash, leaf, stem and root bark were tested at different concentrations of 0, 1, 2, 3, 4 and 5%. One hundred per cent mortality of adult beetles was achieved at all concentrations within 72?h of treatment with extracts except in wood ash at 1% concentration, but they were significantly different (p?<?0.05) from the controls. All the extracts were still able to cause high beetle mortality after one, two and three months of cowpea storage at high concentrations (4 and 5%) except wood ash, although there was a slight decrease in mortality during the period of storage. All the extracts significantly (p?<?0.05) reduced oviposition and adult emergence of C. maculatus when compared with the controls although the reduction was higher at 5% concentration than others. Adult beetle emergence was completely prevented at higher concentrations (4 and 5%) except in wood ash. Both oviposition and adult emergence increased during the months of storage probably because of the slight reduction in the effectiveness of the extracts. The root bark extract was much more effective in reducing oviposition and adult emergence than others throughout the period of storage. The plant extracts of N. laevis was able to protect the cowpea seeds from damage and prevent weight loss. Cowpea seed damage and weight loss was significantly more (p?<?0.05) in the controls than other for the three-month duration probably because of the more adult emergence. The extracts from N. laevis did not adversely affect the germination of the protected seeds and seed germination ranged from 86.7 to 100%. It has been shown in this study that the extracts of N. laevis were effective against C. maculatus in cowpea although the root bark extract seemed to be the most effective while the wood ash extract was least effective. Their effectiveness, however, slightly decreased during the period of storage. N. laevis could be incorporated into pest management of stored cowpeas since the products are ecologically safe.     

Sublethal effects of chronic exposure to chlorpyrifos or imidacloprid insecticides or their binary mixtures on Culex pipiens mosquitoes

Mosquitoes represent one of the most significant threats to human and veterinary health throughout the world. Consequently, improving strategies for the control of mosquitoes is essential. In the present study, juvenile Culex pipiens (Diptera: Culicidae), the common house mosquito, are chronically exposed to sublethal concentrations of chlorpyrifos (20% of LC₅₀) and imidacloprid (5% of LC₅₀), both separately and as a mixture. Developmental time, the emergence rate of adults and the expression of five selected genes involved in detoxification and resistance to pesticides are assessed. To assess the effects on oviposition choice, gravid females are forced to oviposit into cups containing water with added chlorpyrifos, imidacloprid or a mixture of both. The time required for the development of second‐ and third‐instar larvae is observed to differ significantly between treatments. Adults of C. pipiens fail to emerge from larvae hatched in both imidacloprid and the binary mixture. The expression of the four quantified detoxification genes differs significantly in third‐larval instars exposed to chlorpyrifos and/or imidacloprid compared with controls. Gravid females also fail to lay eggs on water to which either of the insecticides or the binary mixture is added, although they do lay eggs in cups containing water only. Chronic exposure to sublethal concentrations of chlorpyrifos or imidacloprid has significant adverse effects on development and thus the reproductive fitness of C. pipiens and, accordingly, could be used in the population control of these mosquitoes.     

Observations on the development of unusual melanization of leopard frog ventral skin

The ontogeny of ventral pigmentation of two species of leopard frog, Rana pipiens and R. chiricahuensis, was examined by light microscopy and transmission electron microscopy to reveal how the unusual melanistic ventral pigmentation of R. chiricahuensis is achieved at the cellular level. Ventral skin of R. pipiens is always white. Ventral skin of adult R. chiricahuensis is white when frogs are background-adapted to a white substrate, but ventral skin becomes nearly as dark colored as the dorsal skin when frogs darken in response to a black background. Skin samples from tadpoles of both species, newly metamorphosed frogs, and adult frogs were analyzed for chromatophore composition and distribution. Ventral skin of R. pipiens larvae, newly metamorphosed frogs, and adults and of R. chiricahuensis larvae was white due to abundant iridophores and no melanophores. Melanophore density in the ventral integument of R. chiricahuensis was 9.1 ± 2.8/mm² in newly metamorphosed frogs and 87.0 ± 4.8/mm² in adult frogs. Pigment within ventral melanophores migrated during physiological color change during background adaptation. © 1993 Wiley-Liss, Inc.     

Landscape genetics reveal broad and fine‐scale population structure due to landscape features and climate history in the northern leopard frog (Rana pipiens) in North Dakota

Prehistoric climate and landscape features play large roles structuring wildlife populations. The amphibians of the northern Great Plains of North America present an opportunity to investigate how these factors affect colonization, migration, and current population genetic structure. This study used 11 microsatellite loci to genotype 1,230 northern leopard frogs (Rana pipiens) from 41 wetlands (30 samples/wetland) across North Dakota. Genetic structure of the sampled frogs was evaluated using Bayesian and multivariate clustering methods. All analyses produced concordant results, identifying a major east–west split between two R. pipiens population clusters separated by the Missouri River. Substructuring within the two major identified population clusters was also found. Spatial principal component analysis (sPCA) and variance partitioning analysis identified distance, river basins, and the Missouri River as the most important landscape factors differentiating R. pipiens populations across the state. Bayesian reconstruction of coalescence times suggested the major east–west split occurred ~13–18 kya during a period of glacial retreat in the northern Great Plains and substructuring largely occurred ~5–11 kya during a period of extreme drought cycles. A range‐wide species distribution model (SDM) for R. pipiens was developed and applied to prehistoric climate conditions during the Last Glacial Maximum (21 kya) and the mid‐Holocene (6 kya) from the CCSM4 climate model to identify potential refugia. The SDM indicated potential refugia existed in South Dakota or further south in Nebraska. The ancestral populations of R. pipiens in North Dakota may have inhabited these refugia, but more sampling outside the state is needed to reconstruct the route of colonization. Using microsatellite genotype data, this study determined that colonization from glacial refugia, drought dynamics in the northern Great Plains, and major rivers acting as barriers to gene flow were the defining forces shaping the regional population structure of R. pipiens in North Dakota.     

Water diffusion and water permeation in eggs from three Anuran species

Water exchange has been studied in ovarian and body cavity eggs of three Anuran species (Rana esculenta, R. pipiens and R. temporaria) with the isotope exchange method using the automatically recording diver balance. In order to estimate the rate of water diffusion in egg cytoplasm (D), a factor required to determine the rate of water permeation (= the exchange coefficient, E), three different chemical treatments (digitonin, ethanol and formaldehyde) have been used to remove the surface barrier to water flow. The obtained mean value of D, 5 × 10^?6 cm² sec^?1, has been accepted as a close approximation of water diffusion in the egg cytoplasm. This value has been used to determine the exchange coefficients in the egg types of the above mentioned species. Comparisons have been made between the calculated values of E both within and between species, and some suggestions have been made concerning the influence of microvilli on the rate of water exchange. Although, on the average, water exchange proceeds at a greater rate in ovarian than in body cavity eggs, a measurable diffusion barrier at the surface can be demonstrated.     

Controlling Culex pipiens: antagonists are more efficient than a neonicotinoid insecticide

Species vulnerability to pesticides depends on physiological sensitivity, the potential to recover, and the ecological context. We assessed the vulnerability of the mosquito Culex pipiens to a repeated treatment with thiacloprid in outdoor microcosms with and without antagonists (competitive and predatory invertebrates). Microcosms were treated repeatedly (three times) with thiacloprid at a concentration of 0.1, 1, or 10 µg/liter. In microcosms without antagonists, the abundance of Cx. pipiens larvae decreased moderately after the second and the third exposures to 10 µg/liter thiacloprid. In microcosms with antagonists, the abundance of Cx. pipiens larvae declined to approximately zero in the control group and the low concentration treatments during the five weeks of observation. By contrast, the abundance of Cx. pipiens larvae temporarily increased at 10 µg/liter thiacloprid after the second and third contamination. We explained this positive effect on the development of Cx. pipiens because of the decrease in competition due to the elimination of sensitive antagonists combined with the high recovery potential of Cx. pipiens. Based on these results, natural antagonists must be supported for the sustainable control of mosquitoes.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Cross-pathogenicity of Rhizoctonia solani strains on pasture legumes in pasture-crop rotations

In Australia, in the past, pasture legumes were rotated mainly with cereals, but increasingly these rotations now involve pasture legumes with a wider range of crops, including legumes. This increasing frequency of the leguminous host in the rotation system may be associated with increased root rots in legumes in the current pasture-crop rotations. The primary aim of this study was to see whether the pathogenicity on pasture legumes of strains of Rhizoctonia solani sourced from lupins and cereals (common crops in rotation with pastures) is associated with increased incidence of root rots in pasture legumes in the disease conducive sandy soils of the Mediterranean regions of southern Australia. The second aim was to determine sources of resistance among newly introduced pasture legumes to R. solani strains originating from rotational crops as this would reduce the impact of disease in the pasture phase. Fifteen pasture legume genotypes were assessed for their resistance/susceptibility to five different zymogram groups (ZG) of the root rot pathogen R. solani under glasshouse conditions. Of the R. solani groups tested, ZG1–5 and ZG1–4 (both known to be pathogenic on cereals and legumes) overall, caused the most severe root disease across the genotypes tested, significantly more than ZG6 (known to be pathogenic on legumes), in turn significantly >ZG4 (known to be pathogenic on legumes) which in turn was >ZG11 (known to be pathogenic on legumes including tropical species). Overall, Ornithopus sativus Brot. cvs Cadiz and Margurita, Trifolium michelianum Savi. cvs Paradana and Frontier and T. purpureum Loisel. cv. Electro showed a significant level of resistance to root rot caused by R. solani ZG11 (root disease scores ≤1.2 on a 1–3 scale where 3 = maximum disease severity) while O. sativus cvs Cadiz and Erica showed a significant level of resistance to root rot caused by R. solani ZG4 (scores ≤1.2). O. compressus L. cvs Charano and Frontier, O. sativus cv. Erica, and T. purpureum cv. Electro showed some useful resistance to root rot caused by R. solani ZG6 (scores ≤1.8). This is the first time that cvs Cadiz, Electro, Frontier, Margurita and Paradana have been recognised for their levels of resistance to root rot caused by R. solani ZG11; and similarly for cvs Cadiz and Erica against ZG4; and for cvs Charano, Erica, and Electro against ZG6. These genotypes with resistance may also serve as useful sources of resistance in pasture legume breeding programs and also could potentially be exploited directly into areas where other rotation crops are affected by these R. solani strains. None of the tested genotypes showed useful resistance to R. solani ZG1–4 (scores ≥2.0) or ZG1–5 (scores ≥2.5). This study demonstrates the relative potential of the various R. solani ZG strains, and particularly ZG1–4, ZG1–5, ZG4 and ZG6 to attack legume pastures and pose a significant threat to non-pasture crop species susceptible to these strains grown in rotation with these pasture legumes. Significantly, the cross-pathogenicity of these strains could result in the continuous build-up of inoculum of these strains that may seriously affect the productivity eventually of legumes in all rotations. In particular, when choosing pasture legumes as rotation crops, caution needs to be exercised so that the cultivars deployed are those with the best resistance to the R. solani ZGs most likely to be prevalent at the location.     

Viability of Culex pipiens pipiens eggs affected by nutrition and aposymbiosis

Raisins were a better source of carbohydrate than sucrose for reproduction by autogenous Culex pipiens. A blood meal increased the number of eggs per raft from 49 autogenously to 114. Eggs of aposymbiotic females produced autogenously did not hatch, but 34% of the eggs were viable if the mosquitoes fed on chickens. With repeated blood meals the number of eggs per raft and the rate of embryonation and hatching declined in each successive gonotrophic cycle. In about $\text{1}\text{4}$ of the unhatched eggs of normal females there were no fully developed embryos, while many more of the unhatched eggs of aposymbiotic females contained no evidence of embryonic development. After the fifth blood meal, neither normal nor aposymbiotic insects oviposited. The ovaries of the nulliparous females contained approximately 10% of the potential number of mature terminal oocytes. Proximally in the ovarioles there were dilatations and coiled tracheoles indicating egg resorption. There were fewer parous follicles in aposymbiotic than in normal females. Larval rearing water, i.e., infusion in which larvae had been reared, was more attractive than fresh infusion for oviposition by normal, autogenous mosquitoes. The degree of embryonation of the eggs was lower and the hatching success rate was poorer in fresh infusion than in larval rearing water.     

Optimization of protein-production by the baculovirus expression vector system in shake flasks

Summary Shake flasks were successfully employed for the cultivation of Spodoptera frugiperda (Sf-9) insect cells and for the production of \-galactosidase, a recombinant model protein, utilizing the baculovirus expression vector system. The culture doubling time and maximal cell density were 20 h and 5 × 10⁶ cells/ml respectively. The optimal liquid volumes for flasks rotating at 100 rpm were 25–40% of the flask total volume. Enzyme production (about 600 mg/l) was best at a multiplicity of infection of between 1 and 20 and at a cell density at time of infection of 0.7 × 10⁶ cells/ml. At a rotation speed of 100 rpm, Pluronic F-68 had no effect on growth and enzyme production. Offprint requests to: Y. Shoham