In vitro rearing of the pupal parasitoidBrachymeria intermedia (Hym.: Chalcididae) on artificial diets with and without host components

The pupal parasitoidBrachymeria intermedia (Nees) was reared from the egg to adult stage on artificial media based on commercial meat homogenates for babies (Plasmon®), either with or devoid of host components. Six media containing 80% homogenate and 20% extract ofGalleria mellonella L. pupae were tested first. Two types of homogenates, intended for babies at the beginning of weaning (a) and well on in weaning (b), were utilized. Media were based on beef, veal and chicken meat, 3 on a-homogenates and 3 on b-homogenates. A significantly higher percentage of parasitoids developed as mature larvae on the a- than on the b-homogenate based diets. This was presumably related to the higher protein, carbohydrate, lipid and calorie level of the a- than of the b- homogenates. Diet veal-a produced the best mean adult yield (27.4%). Other two diets based on the veal-a homogenate were then tested. The first, composed of 80% homogenate, 10% host pupal extract, 7% chicken egg yolk, 1.5% yeast extract and 1.5% wheat germ, produced a mean adult yield of 66.7%, similar to that obtained inG. mellonella pupae. On the second medium, devoid of host components and containing 85% veal-a homogenate, 10% chicken egg yolk, 2.5% wheat germ and 2.5% yeast extract, the mean adult yield was 22.5%. In all media, the adults obtained were viable and fecund.     

Rearing of the pupal parasitoid Brachymeria intermedia on veal homogenate-based artificial diets: evaluation of factors affecting effectiveness

The pupal parasitoid Brachymeria intermedia (Nees) was reared from egg to fecund adult on various veal homogenate-based artificial diets. For every replicate 12.12 ml of each diet were used. Four diets were tested first. Two media, one devoid of and one supplemented with 1 ml Galleria mellonella pupal extract, contained 0.19 g wheat germ and 0.19 g yeast extract each. The other two, one added with and the other devoid of host extract, contained 0.38 g yeast extract each and no wheat germ. All diets also contained chicken egg yolk (1.1 and 0.8 ml in the diets without and with host extract, respectively). The amount of yeast extract was seen to have no significant effect on any of the developmental parameters considered. The replacement of wheat germ with yeast extract was therefore not convenient, considering that the former is far more economical than the latter. Pupal extract was instead found to have a significant effect on pupal and adult yields. The highest adult yield (= 53.2%) was obtained on the diet supplemented with 0.38 g yeast extract and containing host pupal extract. A further four media, each comprising a different kind of material derived from G. mellonella, were subsequently tested. Adult yields were such as to suggest the possibility of replacing pupal with larval extract in the diets as the latter is easier to prepare since there are no cocoons to be removed. In contrast, when the diets were supplemented with larval or pupal homogenate, adult yields dramatically dropped. When B. intermedia was reared in groups rather than individually, most larvae died before attaining maturity. Only two parasitoids, in two different replicates, emerged as adults.     

Growth-promoting effects of ecdysteroids and juvenile hormone on in vitro development of the larval endoparasitoid,Venturia canescens (Hymenoptera: Ichneumonidae)

We previously reported that lipophorin, fetal bovine serum (FBS), and 20-hydroxyecdysone (20-HE) are essential for the development of the larval endoparasitoid Venturia canescens larvae in vitro. The present study was undertaken to determine the optimal concentrations of those three substances in the MGM-450 medium, and to examine the hormonal effects of ecdysteroids and juvenile hormone (JH) on the development of the parasitoid larvae in vitro. When the culture was started with embryos at the post-germband stage, concentrations of 3 mg/ml of lipophorin and 20% of FBS were most suitable for the development of the parasitoid. The growth-promoting effect of 20-HE increased in a concentration-dependent manner and peaked at a concentration of 1 &mgr;g/ml. Excess concentration led to malformations of the larvae. Three other ecdysteroids, ecdysone, 2-deoxy-20-hydroxyecdysone, and polypodine B had the same effect, although their activity was lower than that of 20-HE. Cholesterol had no effect; most larvae failed to develop. When the medium was supplemented with JH, the duration of the developmental period was significantly shortened, but this hormone was not found to be essential.     

Comparison of the efficacy of egg yolk extract and horse serum for growth of Mycoplasma pneumoniae

The usefulness of chicken egg yolk extract as a substitute for horse serum in culture media for Mycoplasma pneumoniae was investigated. As a growth-supporting factor in the growth medium for M. pneumoniae and some other mycoplasmal species, the primary isolation medium for M. pneumoniae, and the metabolism-inhibition test medium for diagnosis of M. pneumoniae infection, egg yolk extract may be an excellent substitute for horse serum. The particular superiority of egg yolk extract to horse serum is that egg yolk extract, unlike horse serum, did not show any inhibitory effect on the growth of M. pneumoniae.     

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.     

Chicken egg yolk-supplemented medium and the serum-free growth of normal mammalian cells

Summary Supplementation of tissue culture medium with chicken egg yolk can support the proliferation of low density bovine vascular and corneal endothelial cells and vascular smooth muscle cells maintained on basement lamina-coated dishes. The optimal growth-promoting effect was observed at concentrations of 7.5 to 10% egg yolk (vol/vol). The average doubling time of bovinn vascular endothelial cells during their logarithmic growth phase when exposed to egg yolk-supplemented medium was longer than that of their counterparts grown in serum-supplemented medium (21 versus 15 h, respectively). Cultures grown in egg yolk-supplemented medium on basement lamina-coated dishes could be serially passaged, but their in vitro life span (15 generations) was less than that of serum-grown cultures (50 generations). The egg white was devoid of any grwoth-promoting activity. This work was supported by Grants HL 20197 and HL 23678 from the National Institutes of Health, Bethesda, MD.     

Entomogenous fungus Nomuraea rileyi inhibits host insect molting by C22-oxidizing inactivation of hemolymph ecdysteroids

The entomogenous fungus Nomuraea rileyi reportedly secretes a proteinaceous substance inhibiting larval molt and metamorphosis in the silkworm Bombyx mori. We studied the possibility that N. rileyi controls B. mori development by inactivating hemolymph molting hormone, ecdysteroids. Incubation of ecdysone (E) and 20-hydroxyecdysone (20E) in fungal-conditioned medium resulted in their rapid modification into products with longer retention times in reverse-phase HPLC. Each modified product from E and 20E was purified by HPLC, and identified by NMR as 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone. Some other ecdysteroids with a hydroxyl group at position C22 were also modified. Injection of the fungal-conditioned medium into Bombyx mori larvae in the mid-4th instar inhibited larval molt but induced precocious pupal metamorphosis, and its injection into 5th instar larvae just after gut purge blocked pupal metamorphosis. In hemolymph of injected larvae, E and 20E disappeared and, in turn, 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone accumulated. These results indicate that N. rileyi secretes a specific enzyme that oxidizes the hydroxyl group at position C22 of hemolymph ecdysteroids and prevents molting in B. mori larvae.     

Wax moth,Galleria mellonella,high density lipophorin receptor: alternative splicing,tissue-specific expression,and developmental regulation

A lipophorin (Lp) receptor cDNA from the fat body of Galleria mellonella (Lepidoptera) was cloned and sequenced. This is the first result in this order, Lepidoptera. It showed the pattern of the VLDL receptor belonging to the LDL receptor family. Sequence homology with other Lp receptors in insects, Locusta migratoria and Aedes aegypti, was 70 and 61%, respectively and each domain was highly conserved. Polyclonal anti-Lp receptor antibody prepared against expressed Lp receptor fragment between ligand binding domain and EGF-precursor homology domain (R305-D549 of amino acid residues) specifically detected the Lp receptor. Through immuno-blotting, the Lp receptor of larval fat body has an approximate molecular mass of about 97 and 110 kDa under non-reducing and reducing conditions, respectively. This result was in agreement with that of the ligand-blotting. The variant Lp receptors were expressed in the fat body of G. mellonella; one is an Lp receptor which lacks 84 bp of O-linked sugar domain and the other is a full length form of the Lp receptor. Both forms were detected by the polyclonal anti-Lp receptor antibody. The Lp receptor from the fat body of G. mellonella was differently expressed depending on the tissue and the developmental stage with specific abundance in prepupal stage. A steroid hormone, 20-hydroxyecdysone (20-HE) plays a crucial role in insect development. With regards to this conception, day 1-2 last instar larvae were treated with 20-HE and drastic induction of the Lp receptor was observed 48 h after treatment. It was also observed that cholesterol caused an induction of the Lp receptor.     

Growth of an ovarian cell line of Galleria mellonella and its response to immune-inducing factors

Antibacterial proteins are produced in the reproductive tracts of some insect species. The advent of a pupal ovarian cell line of the lepidopteran Galleria mellonella offered an opportunity for exploring the use of ovarian tissue culture to induce antimicrobial proteins in lieu of the larvae. The ovarian cell growth rates and cell yields were maximized by adjusting Grace's medium to pH 6.5, adding 15% (v/v) qualified heat-inactivated fetal calf serum, and lowering the sucrose concentration to 9.3 g/L. Five cell forms and biochemical profiles of the collective cell types were analyzed throughout the culture growth cycle. The final modified culture medium did not affect morphogenesis, whereas it increased the culture growth rate by 50% and the final cell yield threefold. The molting and immunoprotein-inducing hormone, 20-hydroxyecdysone, increased culture growth rate and altered the levels of cell types A and D. Neither 20-hydroxyecdysone nor the larval immunizing agents, apolipophorin-III or Bacillus subtilis, in combination or alone, induced antibacterial activity. The bacterium did induce immunity in both larval and adult stages.     

Experimental manipulation of egg quality in chickens: influence of albumen and yolk on the size and body composition of near-term embryos in a precocial bird

The importance of avian egg components in the determination of hatchling size and quality has yet to be fully evaluated. In the first experiment, 20% of the albumen and/or the yolk was removed from chicken eggs to determine the impact of each egg component on metabolism and various size measures in near-term embryos. Results show that metabolic rate, dry body mass, and internal organ mass are largely independent of egg composition. Removal of albumen resulted in a decrease in wet body mass corresponding to decreases in water content in the body and the yolk sac, and decreased tibiotarsus length. Removal of yolk resulted in no change in body mass, but decreases in both wet and dry yolk sac mass. In a second experiment, removal of 15% of either egg component led to reductions in hatchling mass similar to those observed in whole near-term embryos. Albumen, as the primary source of water in the egg, is the primary determinant of hatchling size and may influence hatchling success through size-related limiting factors. Differences in yolk content may influence neonatal quality as a nutritional supplement, but seem not to result in greater tissue formation during embryonic development. Accepted: 2 September 1997     

Separation of Viable Rickettsia typhi from Yolk Sac and L Cell Host Components by Renografin Density Gradient Centrifugation

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.     

In Vitro Rearing of the Parasitoid Exorista larvarum (L.) (Diptera: Tachinidae) on Meat Homogenate-Based Diets

The tachinid Exorista larvarum (L.), a polyphagous gregarious larval endoparasitoid of Lepidoptera, was reared from egg to fecund adult on media containing commercial meat homogenates for babies as the main ingredient. Four media, each containing a diverse homogenate supplemented with extract of Galleria mellonella L. pupae, were tested first. Despite the difference in nutrient content, the kind of homogenate did not significantly affect the adult yields (30.2 to 40.7%) or puparial weights. Two other diets free of host materials (I and II) were then tested. Both were based on Gerber veal homogenate combined with different amounts of yeast extract and chicken egg yolk and were supplemented with wheat germ (I) or saccharose (II). Adult yields (28.7 to 32.7%) and puparial weights did not differ significantly between the two diets. Fly longevity and fecundity of the females obtained on diet I were comparable to those of the females emerged from puparia formed in G. mellonella larvae. Male and female puparial weights were, however, higher and development times longer on the diet than in the host.     

Quail Egg Yolk: A Novel Cryoprotectant for the Freeze Preservation of Poitou Jackass Sperm

For many years, attempts have been made to establish a sperm bank for the Poitou jackass population which is threatened with extinction. Unfortunately, no cryopreservation technique has ever been described for spermatozoa of this species. In an attempt to find a suitable technique, we studied the relative effectiveness of chicken egg yolk and quail egg yolk in preserving the motility and characteristics of movement of Poitou jackass spermatozoa during the freezing–thawing process. Semen was diluted to 60 × 10⁶sperm/ml in a preservation medium containing 4% (v/v) glycerol with 0, 2, 5, 10, 15, or 20% (v/v) of chicken or quail egg yolk. The chemical composition of these two eggs was compared. Effects were assessed using an automated analyzer which measured curvilinear velocity (VCL), straight line velocity (VSL), and the velocity of the average path. Linearity was defined as VSL/VCL × 100. The amplitude of the lateral head displacement was also measured. It was found that after the freeze–thaw process, quail egg yolk improved the percentages of motile and progressively undulating spermatozoa and the movement characteristics compared with chicken egg yolk. The optimal concentration of quail egg yolk was 10%. The general composition of the two types of egg yolk were similar, but quail egg yolk contained significantly more phosphatidylcholine, less phosphatidylethanolamine, and a smaller ratio of polyunsaturated to saturated fatty acids than chicken egg yolk. The improvement of motility for frozen–thawed Poitou jackass spermatozoa using frozen–thawed quail egg yolk compared to chicken egg yolk may be due to the differences in composition of the two yolks.     

Lipophorin as a yolk precursor in Hyalophora cecropia: uptake kinetics and competition with vitellogenin

Vitellogenic follicles of Hyalophora cecropia were incubated in metabolically radiolabeled, high-density lipophorin isolated from pharate adult hemolymph by KBr density gradient centrifugation. The follicles transferred this probe from the incubation medium to the cortical yolk spheres in the oocyte by an energy-dependent and saturable mechanism. Vitellogenin and high-density lipophorin competed with each other for uptake, and are therefore concentrated by the follicle with a common mechanism. Microvitellin and lipophorin, in contrast, did not compete for uptake. The K(uptake) for the accumulation of high-density lipophorin was substantially higher than the value estimated earlier for vitellogenin (133 microM vs. 18 microM). This relationship helps explain why the shared concentrating mechanism does not deplete the lipid transport capacity of the hemolymph, and how a low vitellogenin: lipophorin molar ratio in the hemolymph yields a high ratio in the mature egg.     

Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens

We previously reported the production of human erythropoietin (hEpo) using genetically manipulated (GM) chickens. The recombinant hEpo was produced in the serum and egg white of the GM chickens, and the oligosaccharide chain structures of the serum-derived hEpo were more favorable than those of the egg white-derived hEpo. In the present study, a retroviral vector encoding an expression cassette for a fusion protein of hEpo and the Fc region of human immunoglobulin G (hEpo/Fc) was injected into developing chicken embryos, with the aim of recovering the serum-derived hEpo from egg yolk through the yolk accumulation mechanism of maternal antibodies. The GM chickens that hatched stably produced the hEpo/Fc fusion protein not only in their serum and egg white, but also in the egg yolk as expected. Lectin blot analyses revealed that significant amounts of the oligosaccharide chains of hEpo/Fc produced in the serum and eggs of GM chickens terminated with galactose, and that the oligosaccharide chains of the serum- and yolk-derived hEpo/Fc incorporated sialic acid residues. Moreover, biological activity assessment using Epo-dependent cells revealed that the yolk-derived hEpo/Fc exhibited a comparable performance to the serum- and CHO-derived hEpo/Fc. These results indicate that transport of Fc fusion proteins from the blood circulation to the yolk in chickens represents an effective strategy for the production of pharmaceutical glycoproteins using transgenic chicken bioreactors.     

Male-grown eggs in Hyalophora: Deficient follicle cell secretion as well as protein and lipid yolk deposition

Ovaries transplanted to male Lepidoptera during late larva or pupal stages produce smaller and fewer chorionated eggs than those remaining in place or transplanted to other females. Small size is shown here in Hyalophora cecropia to result not only from a lack of vitellogenic hemolymph proteins but also from dysfunction of the follicular epithelium. Several aspects of egg formation can proceed normally in the male environment, including RNA deposition by the nurse cells, the conversion of lipophorin to a very high density form as the oocyte endocytoses it, and the customary period of osmotic swelling between the end of yolk deposition and the beginning of chorion formation. But as would be expected, male-grown eggs lack vitellogenin and contain very little microvitellogenin. They also contain lower than normal amounts of lipophorin, which is related to the male's poor ability to replace this protein as the oocyte removes it from the hemolymph. A low phospholipid content can be attributed to the absence of vitellogenin and a low triglyceride droplet content to the shortage of lipophorin. Two other deficiencies, however, could not be directly explained by the low levels of vitellogenic hemolymph proteins: paravitellogenin and chorion, both secretions of the follicle cells, are deposited in significantly reduced amounts. Males of this species, in addition to lacking sufficient vitellogenic proteins and lipids in their hemolymph, are thus unable to fully support the secretory activities of the follicle cells.     

A comparison of the protective action of added egg yolks from five avian species to the cryopreservation of bull sperm

Cryopreservation of domestic animal sperm has been widely used for artificial insemination (AI), and egg yolk is one of the most commonly used cryoprotectants during the freezing-thawing process. The objectives of this study were to compare the effectiveness of egg yolk from five avian species (domestic chicken, domestic duck, domestic goose, Japanese quail or domestic pigeon) and to optimize the concentration of egg yolk on the cryopreservation of bull sperm in terms of frozen-thawed sperm progressive motility and viability. The results were two-fold. First, they showed that pigeon egg yolk provided the best cryoprotective effects on the cryopreservation of bull sperm, compared with egg yolk of chicken, quail, goose or duck. Second, the best concentration of pigeon egg yolk in extender was 20% during cryopreservation among five concentrations of 5, 10, 20, 30 or 40%. The results suggest that pigeon egg yolk could be used as an alternative to chicken egg yolk in extender but requires further testing in fertility trials.     

20-Hydroxyecdysone accelerates the flow of cells into the G1 phase and the S phase in a male accessory gland of the mealworm pupa (Tenebrio molitor)

The cells of the bean-shaped accessory glands of mealworms proliferate through the first 7 days of the 9-day pupal stage. Immediately after larval-pupal ecdysis, 25-27% of the cells were in the G1 phase, 60-65% were in the G2 phase, and the balance were in S phase. Over the first 4 days of normal development, the S fraction gradually increased, to reach its highest level in the mid-pupa at the time of the major ecdysteroid peak (Delbecque et al., 1978). Thereafter, the S fraction declined until over 95% of the cells had accumulated in G2 on Day 8. When 0-day pupal glands were explanted into Landureau's S-20 medium for 6 days, the G1 fraction remained fairly constant (25-30%) while S and the G2 fractions fluctuated. On the first day in vitro, the G2 fraction declined and the S fraction rose. On the second day in basal media, the S fraction fell and G2 rose correspondingly until 70% of the cells reached G2 when cycling stopped on the third day. With addition of 20-hydroxyecdysone to 0-day cultures, the S fraction increased quite sharply. It remained large for all 6 days of the experiment in the continuing presence of hormone. A 1-day pulse of hormone produced a transient increase in S. We blocked cell cycling with hydroxyurea in a stathmokinetic experiment and showed that 20-hydroxyecdysone accelerated the flow of cells from the G2 phase to the G1 phase by 2.5-fold. An increase in the G1 fraction was detected within 10 hr of hormone administration and the effect was dose-dependent with an ED50 of 5 X 10(-7) M for 20-hydroxyecdysone. We conclude that 20-hydroxyecdysone acts at a control point in the G2 phase. Incubation of the glands with 20-hydroxyecdysone for only 30-60 min followed by washout stimulated the flow from G2 to G1 and the effect persisted after transfer of the tissues to hormone-free media. Dose-dependent stimulation also occurred with ponasterone A (ED50 3 X 10(-9] but not with cholesterol.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Adsorptive endocytosis of vitellogenin,lipophorin, and microvitellogenin during yolk formation in Hyalophora

Yolk in Hyalophora cecropia is a mixture of proteins that are derived from the extracellular medium. We have measured for five of these proteins the number of moles deposited in each egg, the molarity of their precursors in the hemolymph at a midpoint in vitellogenesis (day 18 of adult development), and the degree to which they are concentrated by the oocyte, relative to inulin. The proteins were isolated by gel permeation and ion exchange chromatography and used to generate antibodies in rabbits. Preliminary studies established that yolk proteins are essentially quantitatively extractable in media suitable for measuring antigen concentrations by precipitation with antibodies and that yolk and hemolymph forms of the five proteins have, effectively, the same antibody-binding specificities as the isolated standards. Content per egg was about 900 pmol for vitellogenin, 600 pmol for microvitellogenin, and 300 pmol for lipophorin. By contrast, two hemolymph storage hexamers, arylphorin and a flavoprotein, occurred at less than 3 pmol per egg. In principle, yolk precursors are taken in both as solutes in the fluid phase of the endocytotic vesicles and as ligands adsorbed to vesicle membranes. Measurements of inulin uptake indicated that fluid phase endocytosis could account for only 4% of vitellogenin, 1% of microvitellogenin, and 15% of lipophorin in the yolk, when hemolymph precursors are at their day 18 concentrations. By the same comparison, arylphorin and flavoprotein appear to be excluded from the yolk, relative to inulin.