Evidence for a calcium-binding site on the eggshell of Globodera rostochiensis with a role in hatching

Two inhibitors of hatching in Globodera rostochiensis, ruthenium red and lanthanum, have been shown to bind to the eggshell using the techniques of microdensitometry for ruthenium red and X-ray microanalysis for lanthanum. Neither inhibitor penetrated or adhered to unhatched or hatched viable juveniles. Scatchard analysis for binding of lanthanum and ruthenium red to eggshells gave dissociation constants (K) of K_La 32.5 ± 14.0 μM and K_Rured 33.5 ± 5.0 μM respectively. Both values are within the 95% fiducial limits of those shown to cause 50% inhibition of hatch in previous work. Pretreatment with sodium hypochlorite separated an outer part of the eggshell from an inner region which exclusively bound ruthenium red. It is the inner lipoprotein layer that is believed to include the membranes controlling the permeability of the tylenchid eggshell. The rate of binding of ruthenium red was similar for intact and isolated eggshells with 50% binding occurring after 6.11 ± 0.91 min and 4.95 ± 2.38 min but the latter gave a significantly higher maximum binding suggesting that rupture of the eggshells made available additional binding sites on their inner surface. The binding of ruthenium red to the eggshells was pH dependant over most of the range pH 2.8–8.5 with 50% binding, given with its standard deviation, occurring at pH 5.75 ± 0.85. Competitive binding of lanthanum influenced the binding of ruthenium red to the eggshells from which Scatchard analysis gave K_la of 176 ± 79 μM. Similarly, calcium influenced the binding but this caused a biphasic plot with high and low affinity binding sites of K“_ca of 0.423 ± 1.16 μm and K‘_ca of 1078 ± 462 μM. The existence of a high affinity site for calcium that also binds ruthenium red, suggests that the eggshell membrane includes a calcium binding glycoprotein as found in some other receptor mechanisms.     

Nematodirus battus: Permeability changes,calcium binding,and phosphorylation of the eggshell during hatching

Eggshells of Nematodirus battus leaked trehalose 4 hr after being stimulated to hatch, and became permeable to trypan blue at their poles; 80% of eggs were stained blue 24 hr later. Exogenous application of ruthenium red significantly inhibited chill- and sodium fluoride-stimulated hatching, 50% hatch inhibition occurring in 44.67 ± 2.2 and 8.5 ± 1.5 μM, respectively. Lanthanum chloride, however, was not as inhibitory as ruthenium red on fluoride-stimulated hatching, 50% occurring at 31.60 ± 1.25 μM. A Scatchard plot of the competitive binding of ruthenium red to eggshells demonstrated a high-affinity binding site for calcium, K_Ca′ = 1.92 μM and a second, low-affinity site, K_Ca′ = 1169.60 μM. Ruthenium red binding was significantly reduced by several enzymes, e.g., EGTA-buffered trypsin reduced binding by 73%. Radioiodinated concanavalin A also bound competitively to the eggshells in the presence of α-d-glucosyl-α-d-glucopyranoside and α-methyl-d-mannopyranoside. Eggshells incorporated phosphorus-32 from ATP after chilling or on exposure to sodium fluoride; gel filtration of solubilized homogenates of these samples showed that two proteins were radiolabelled with molecular weights of 38 × 10³ and 8 × 10³ Da, respectively. This phosphorylation was inhibited by N-ethylmaleimide, which also prevented hatching.     

Evidence for the involvement of calcium in the hatching of Globodera rostochiensis

Low concentrations of ruthenium red and lanthanum chloride inhibited hatching of juveniles from eggs in cysts of Globodera rostochiensis in potato root diffusate. Pro-bit analysis for 31 cysts at seven concentrations of ruthenium red showed that 50% inhibition with 95% fiducial limits occured at 47 ± 23 μm; a similar value of 59 ± 14 μm was obtained using eggs removed from cysts. Results for 10 to 20 cysts at six concentrations of lanthanum chloride suggested a somewhat higher value for 50% inhibition of 110 ± 83 μM. In contrast hatching of eggs in cysts of Heterodera schachtii in water was unaffected by 5 ITIM lanthanum chloride and 625 μM ruthenium red, concentrations which cause over 90% inhibition of hatch in G. rostochiensis.
Two calcium ionophores synergised hatching of a 1971 population of G. rostochiensis in dilute diffusate. Optimal concentrations of 2 μM for A23187 and 10 μM for BrX537A increased the hatch from 17 ± 3–6 juveniles/cyst to 114 ± 44 juveniles/cyst and 138 ± 40 juveniles/cyst respectively. Ionophores in the absence of diffusate hatched very few eggs of this population but caused a greater hatch in a second (1975) population which gave a high hatch in water of 43 ± 10 juveniles/cyst. This was increased by A23187 to 181 ± 41 juveniles/cyst. The results with both the inhibitors and the ionophores suggest that hatching in G. rostochiensis might be a calcium-mediated process.     

External incubation alters the composition of squamate eggshells

Eggshells of the skink, Eumeces fasciatus, and of the rough green snake, Opheodrys vernalis, contain 28-40% of total shell mass as calcium at oviposition. After incubation, both calcium concentration and content were reduced 17-41%. Protein accounted for 33-65% of shell mass in Eumeces and Opheodrys eggshells at oviposition. Reductions in protein concentration and content from 22-86% following incubation were observed. Comparing amino acid compositions of eggshells collected following oviposition with those collected from the same clutch after hatching reveals similarities among all eggshells except those of Eumeces from Michigan which lack desmosine and isodesmosine. Concentrations of desmosines in eggshells from Missouri and Michigan Eumeces appeared to decrease following incubation by 49.7% and 12.2%, respectively. Although neither the mechanisms involved in apparent losses of calcium and protein, nor the fate of the mobilized materials are known, possible pathways for mobilization of eggshell constituents are outlined and their pertinence to the physiology of squamate eggs and the evolution of viviparity in reptiles is suggested.     

Possible involvement of calcium ions in the hatching of Schistosoma mansoni eggs in water

The possibility of involvement of calcium ions in the hatching of Schistosoma mansoni eggs in water is described. The hatching of S. mansoni eggs under low osmotic pressure was partially inhibited by EGTA (5 mM), lanthanum chloride (1-5 mM), and ruthenium red (0.1-1 mM). The reagents used in these experiments were not toxic to the eggs however, because miracidia hatched normally when the reagents were removed.     

Structure of shells from eggs of kinosternid turtles

Shells from eggs of five species of kinosternid turtle (Sternotherus minor, Kinosternon flavescens, K. baurii, K. Hirtipes, and K. alamosae) were examined with light and scanning electron microscopy. Except for possible differences among species in thickness of eggshells, structure of shells from all eggs was similiar. In general, kinosternid turtles lay eggs having a rigid calcareous layer composed of calcium carbonate in the form of aragonite. The calcareous layer is organized into individual shell units with needlelike crystallites radiating from a common center. Most of the thickness of the eggshell is attributable to the calcareous layer, with the fibrous shell membrane comprising only a small fraction of shell thickness. Pores are found in the calcareous layer, but they are not numereous. The outer surface of the eggshells is sculptured and may have a thick, organic layer in places. The outer surface of the shell membrane of decalcified eggshells is studded with spherical cores which presumably nucleate growth of shell units during shell formation. The shell membrane detaches from eggs incubated to hatching, carrying with it remnants of the calcareous layer. Such changes in shell structure presumably reflect withdrawal of calcium from the eggshell by developing embryos.     

Differential label‐free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property

Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label‐free MS‐based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.     

Sediment and eggshell interactions: using abrasion to assess transport in fossil eggshell accumulations

Recent excavation at the Late Cretaceous Egg Mountain locality in Montana revealed abundant Spheroolithus eggshell fragments. The depositional history of the fragments is problematic due to the vertical dispersal of eggshell in homogeneous mudstone. The results of previous studies assessing eggshell transport are based on thin, modern eggshell occurring on well-defined horizons. Therefore, the assessment of depositional history required a different method. Chicken (Gallus gallus domesticus) and ostrich (Struthio camelus) eggshells were placed in a tumbler with water and quartz sand for 168–504 h to simulate transport. The resulting wear on these fragments, revealed under scanning electron microscope, was compared to unabraded eggshell. In addition, the modern eggshell was compared to fossil eggshells from a nesting site, crevasse splay and channel deposits, and Egg Mountain. Fossil eggshells from high-energy paleoenvironments display similar edge rounding to modern eggshell placed in the tumbler. In contrast, eggshell from Egg Mountain lacked edge rounding and resembled fossil eggshell from a nesting site and unabraded modern eggshell, suggesting that the Egg Mountain locality represents a parautochthonous assemblage. This study indicates that sediment interaction leaves distinct patterns of abrasion on eggshell, which may be useful for assessing transport of eggshell at fossil localities.     

Fine structure of the subitaneous eggshell of the sessile rotifer Stephanoceros millsii (Monogononta) with observations on vesicle trafficking in the integument during ontogeny

Rotifers that engage in cyclical parthenogenesis produce two types of eggs: subitaneous eggs that hatch as clonal females and meiotic eggs that hatch as haploid males, or if fertilized, as females after a period of diapause (resting eggs). The ultrastructure of resting eggshells is known for some motile species, but there are limited data on subitaneous eggshells, and no data on any eggshells of sessile rotifers. Here, we investigated the ultrastructure of the subitaneous eggshell of the sessile rotifer Stephanoceros millsii and its potential origins of secretion, the maternal vitellarium and embryonic integument. We also explored secretory activity in the larval and adult integuments to determine whether activity changes during ontogeny. The eggshell consists of a single layer with two sublayers: an external granular sublayer apparently derived from the maternal vitellarium, and an internal flocculent sublayer secreted by the embryonic integument that may form a hatching membrane or glycocalyx. Secretory activity remains high in both the larva and adult and appears to be the source of the thickening glycocalyx. Altogether, the subitaneous eggshell of S. millsii is the thinnest among monogonont rotifers. Thin eggshells may have evolved in response to the added protection provided by the mother’s extracorporeal tube.     

The fine structure of the glycocalyx of equine spermatozoa: a high-resolution cytochemical study.

Equine spermatozoa were obtained from ejaculates of young stallions. The seminal plasma was removed and the sperm pellets washed three times with 0-15 M-NaCl solution before final centrifugation at 4500 g for 15 min. The pellets were fixed in a mixture of 2-5% glutaraldehyde in 0-1 M-cacodylate buffer, pH 7-4, with 0-5% Alcian blue and post-fixed in 1% osmium tetroxide with 1% lanthanum nitrate; other samples were treated with ruthenium red. All samples were dehydrated in ascending concentrations of ethanol, embedded in araldite and thin sections examined in an electron microscope. Electron dense deposits of lanthanum were present on the surface plasmalemma of the head, mid-piece and tail of 70% of mature spermatozoa, and similar deposits were seen in ruthenium red-treated samples. No glycocalyx was observed in untreated spermatozoa.     

Characterization of calcium binding to adipocyte plasma membranes.

Calcium binding to adipocyte plasma membranes has been assessed by equilibrium dialysis and by membrane filtration techniques. Calcium binding was specific and saturable, displaying two distinct classes of binding sites. The affinity constants and maximum binding capacities in the presence of 0.1 M KCl were 4.5 X 10(4) M-1 and 1.8 nmol/mg of protein and 2.0 X 10(3) M-1 and 13.7 nmol/mg for the high and low affinity sites, respectively. Bound calcium was totally dissociated in the presence of excess calcium within 11.0 min in two distinct phases corresponding to the two classes of sites. Association and dissociation rate constants for the high affinity sites were 7.7 X 10(2) M-1S-1 and 9.2 X 10(-3S-1 respectively. Free energy changes at 24 degrees were +6.4 kcal mol-1 for the high affinity sites and +4.5 kcal mol-1 for the low affinity sites. The high affinity sites demonstrated a pH optimum of 7.0 whereas the binding to the low affinity sites progressively increased between pH 6.0 and 9.0. Low concentrations of MgCl2 (less than 300 muM) enhanced calcium binding slightly, whereas high concentrations of KCl and MgCl2 were noncompetitive inhibitors of calcium binding. Procaine and ruthenium red had no effect on calcium binding and lanthanum was a poor inhibitor of calcium binding. This represents the first report of calcium binding to adipocyte plasma membranes and the first kinetic analysis of calcium binding to biological membranes. The specificity of this calcium-binding system in adipocyte plasma membranes suggests its importance in cellular bioregulation.     

Mitotic progression in stamen hair cells of Tradescantia is accelerated by treatment with ruthenium red and Bay K-8644

The normally predictable duration of metaphase in stamen hair cells from the spiderwort, Tradescantia virginiana, is shortened significantly by treatment during prometaphase with either ruthenium red or Bay K-8644. Ruthenium red is an inhibitor of Ca2+ translocation and Bay K-8644 is a Ca2+-channel agonist. Their action on mitotic progression appears to involve a rise in the cytosolic Ca2+ level that in turn has a pronounced effect on the duration of metaphase. The timing of addition of ruthenium red for accelerated progression through metaphase is less critical than that for Bay K-8644 which will promote metaphase progression only if added 0 to 12 min after nuclear envelope breakdown. In contrast, ruthenium red can be added at any time from approximately 10 min prior to nuclear envelope breakdown up to 25 min afterward. A reduction of extracellular Ca2+ is sufficient by itself to prolong the duration of metaphase in stamen hair cells, but the duration of metaphase by ruthenium red or Bay K-8644 is significantly shortened in identical solutions with Ca2+ buffered at levels greater than 1 microM. Metaphase progression rates with either agent are independent of changes in extracellular Mg2+ levels. Correlated with the precocious entry into anaphase was rapid formation of the spindle and a marked reduction in spindle rotation during metaphase. Interestingly, we observed a modest increase in the rate of anaphase chromosome separation, but the appearance of cell plate vesicles at the site of incipient cell plate formation occurred normally approximately 19 min after anaphase onset. Similarly, the initial appearance of cell plate vesicles in Bay K-8644 was normal, approximately 19 min after the onset of anaphase. These results further implicate shifts in cytosolic Ca2+ in the regulation of mitotic events.     

Placental calcium provision in a lizard with prolonged oviductal egg retention

A prominent scenario for the evolution of viviparity and placentation in reptiles predicts a step-wise pattern with an initial phase of prolonged oviductal egg retention accompanied by progressive reduction in eggshell thickness culminating in viviparity; calcium placentotrophy evolves secondarily to viviparity. Saiphos equalis is an Australian scincid lizard with a reproductive mode that is uncommon for squamates because eggs are retained in the oviduct until late developmental stages, and the embryonic stage at oviposition varies geographically. We studied calcium mobilization by embryos in two populations with different oviductal egg retention patterns to test the hypothesis that the pattern of nutritional provision of calcium is independent of the embryonic stage at oviposition. Females from one population are viviparous and oviposit eggs containing fully formed embryos, whereas embryos in oviposited eggs of the second population are morphologically less mature, and these eggs hatch several days later. The reproductive mode of this population is denoted as prolonged oviductal egg retention. Yolk provided the highest proportion of calcium to hatchlings in both populations. Eggs of both populations were enclosed in calcified eggshells, but shells of the population with prolonged egg retention had twice the calcium content of the viviparous population and embryos recovered calcium from these eggshells. Placental transfer accounted for a substantial amount of calcium in hatchlings in both populations. Hatchling calcium concentration was higher in the population with prolonged egg retention because these embryos mobilized calcium from yolk, the eggshell and the placenta. This pattern of embryonic calcium provision in which both a calcified eggshell and placentotrophy contribute to embryonic nutrition is novel. The reproductive pattern of S. equalis illustrates that calcified eggshells are compatible with prolonged oviductal egg retention and that viviparity is not requisite to calcium placentotrophy.     

The evolution of size of the uropygial gland: mutualistic feather mites and uropygial secretion reduce bacterial loads of eggshells and hatching failures of European birds

Potentially, pathogenic bacteria are one of the main infective agents against which a battery of chemical and physical barriers has evolved in animals. Among these are the secretions by the exocrine uropygial gland in birds. The antimicrobial properties of uropygial secretions may prevent colonization and growth of microorganisms on feathers, skin and eggshells. However, uropygial gland secretions also favour the proliferation of feather mites that feed on secretions and microorganisms living on feathers that would otherwise reach eggshells during incubation if not consumed by feather mites. Therefore, at the interspecific level, uropygial gland size (as an index of volume of uropygial secretion) should be positively related to eggshell bacterial load (i.e. the risk of egg infection), whereas eggshell bacterial loads may be negatively related to abundance of feather mites eating bacteria. Here, we explore these previously untested predictions in a comparative framework using information on eggshell bacterial loads, uropygial gland size, diversity and abundance of feather mites and hatching success of 22 species of birds. The size of the uropygial gland was positively related to eggshell bacterial loads (mesophilic bacteria and Enterobacteriaceae), and bird species with higher diversity and abundance of feather mites harboured lower bacterial density on their eggshells (Enterococcus and Staphylococcus), in accordance with the hypothesis. Importantly, eggshell bacterial loads of mesophilic bacteria, Enterococcus and Enterobacteriaceae were negatively associated with hatching success, allowing us to interpret these interspecific relationships in a functional scenario, where both uropygial glands and mutualistic feather mites independently reduce the negative effects of pathogenic bacteria on avian fitness.     

Global diversity and adaptations of avian eggshell thickness indices

The amniote eggshell is a fundamental aspect of the embryo life-support system, protecting it from UV light, microbes and mechanical damage, while regulating gas exchange and providing calcium for growth. The thickness of eggshells is highly diverse across modern birds and influences multiple eggshell functions, yet the selective pressures driving eggshell thickness have not been clearly identified. Here, we use a global dataset of avian eggshell thickness indices for 4260 (> 41%) avian species to assess trends in eggshell thickness across the phylogeny, as these indices are strongly correlated with direct measures of eggshell thickness and are non-destructive to the sample. We analysed the dataset within a phylogenetic framework to assess the relative importance of climatic and ecological explanations for variation in eggshell thickness indices. The distribution of avian eggshell thickness indices across species was found to be primarily driven by phylogenetic relatedness, in addition to evolutionary processes that do not match a Brownian model of evolution. Across modern birds, thicker eggshells were more prevalent in species (1) with precocial young, (2) which exhibit a scavenger-based diet, (3) which primarily feed on vertebrates or plants (excluding nectivores, seed and fruit specialists) and (4) which breed in open habitats. Thicker eggshells found in species with precocial young probably enable higher rates of calcium removal for the more advanced embryo development. Excessive light transmission through the shell damages developing embryos, while too little light transmission can impede development. Eggs in shaded habitats experience low light exposure, and thus thinner shells are more prevalent in these environments potentially to increase light transmission through the shell. Overall, variation in eggshell thickness indices appears to be driven largely by phylogeny, with certain life-history traits linked to embryo growth rate, calcium content of their food, and the need to mitigate excessive light transmission through the shell.     

External calcium inhibits the efflux of calcium from isolated retinal rod outer segment disks

Increasing the concentration of calcium in the external buffer flowing past isolated, intact bovine retinal rod outer segment disks immobilized in a flow system reduced the rate of radioactive calcium efflux from within the disks in the dark. We interpret these results as extradiskal calcium acting at an inhibitory binding site to block the calcium efflux. A Scatchard analysis of the external calcium dependence of the efflux yields an apparent dissociation constant of 50 microM, which further suggests that the inhibition is mediated by a specific membrane binding site. The observed inhibition of calcium efflux may represent a functional role for the high-affinity calcium binding site which has been identified by others in previous physical studies of the disk membrane. This external calcium inhibited permeability may explain some of the discrepancies in the reported calcium transport properties of disks. Variations in the external calcium concentration may alter the calcium content of isolated disks, thereby indirectly affecting other transport functions including the measured light-induced release of calcium. No evidence was found for either Na/Ca or Ca/Ca exchange processes across the disk membrane. Lanthanum was even more effective than calcium in inhibiting calcium efflux in the dark. Neither lanthanum nor calcium inhibited the light-induced efflux of calcium from disks, which implies either that light and extradiskal calcium regulate separate permeability processes in the disk membrane or that light greatly reduces the affinity of the inhibitory site for calcium and lanthanum.     

Eggshell coloration is an indicator of dietary calcium in Common Pheasants (Phasianus colchicus)

According to the structural-function hypothesis, the eggshell pigment protoporphyrin, deposited at weak spots, can strengthen the shell structure when calcium is lacking in avian species. However, this hypothesis has not been tested in species that produce pigmented eggs of uniform ground colour without spotting patterns. We tested the structural-function hypothesis using 435 eggs produced on seven calcium diets (0.2–4.5%) given to Common Pheasants Phasianus colchicus, a species that produces a large number of eggs on a low-calcium diet with unspotted eggshells composed of a uniform ground colour of mainly protoporphyrin. We found that pheasants on the lowest calcium diet (0.2%), which had thinner eggshells, produced eggs containing more protoporphyrin-based coloration than four of six other diets, suggesting this species employs pigmentation as ground colour to strengthen eggshells when available calcium is low. Our results provide the first, at least partial support for a structural function for eggshell pigments producing ground colour without spotting in a species that is often calcium-limited. This pattern may be more widespread in other ground-nesting taxa that also produce large numbers of eggs with protoporphyrin-based ground colour and are potentially limited by calcium during breeding.     

Effects of calcium and lanthanum on phosphate efflux from nonmyelinated nerve fibers

Summary Phosphate efflux was measured as the fractional rate of loss of radioactivity from rabbit vagus loaded with radiophosphate. The effects of changes in extracellular calcium and of lanthanum have been investigated. In Locke solution with normal, 0.9mm, calcium and without phosphate, the fractional rate of loss was 1.62×10^–3 min^–1 at 120 min after the beginning of the washing period and fell slowly (9% hr^–1) during washing from 2 to 6 hr. Addition of calcium to the Locke solution produced a transient increase followed by a reversible maintained increase in phosphate efflux. The latter was 40 and 75% above efflux in normal calcium for 20 and 50mm calcium, respectively. Removal of calcium, with or without addition of EGTA, produced only a transient increase in phosphate efflux, with no subsequent maintained change. Addition of low concentrations of lanthanum produced a reversible inhibition of phosphate efflux. Half-maximal inhibition was at 3.5 m lanthanum and appeared to be due to binding of lanthanum to more than one, probably two, sites. Measurements of inhibition by lanthanum at different calcium concentrations did not indicate any competition between calcium and lanthanum. It is suggested that at least a part of phosphate efflux depends on internal calcium and that lanthanum acts by preventing release of phosphate from the phosphate transport mechanism.     

Inhibitory Properties of Ruthenium Amine Complexes on Mitochondrial Calcium Uptake

The recent finding that the inhibition of Ca²⁺-stimulated respiration by ruthenium red is mainlydue to a binuclear ruthenium complex (Ru₃₆₀) present in the commercial samples of the classicalinhibitor ruthenium red (Ying et. al., 1991), showed that this complex is the more potent andspecific inhibitor of the mitochondrial calcium uniporter. This work was aimed to provideinsights into the mechanism by which Ru₃₆₀ and other ruthenium-related compounds inhibitscalcium uptake. Ruthenium red and a synthesized analog (Rrphen) were compared with Ru₃₆₀.The inhibition by this binuclear complex was noncompetitive, with a K _i of 9.89 nM. Thenumber of specific binding sites for Ru₃₆₀ was 6.2 pmol/mg protein. Ruthenium red and Ru₃₆₀were mutually exclusive inhibitors. Bound La³⁺ was not displaced by Ru₃₆₀. Rrphen was theleast effective for inhibiting calcium uptake. The results support the notion of a specific bindingsite in the uniporter for the polycationic complexes and a negative charged region from thephospholipids in the membrane, closely associated with the uniporter inhibitor-binding site.     

Relationship between age and colour of hatched eggshells of Ochlerotatus vigilax (Skuse) (Diptera: Culicidae) on an Australian saltmarsh

Abstract The presence of aedine mosquito eggshells in soil has recently been used to delineate oviposition patterns in coastal wetlands. However, the colour of eggshells may reveal information about the oviposition activity of mosquitoes that is not evident from the density and distribution of eggshells alone. This study describes the rate of colour change of newly hatched eggshells of Ochlerotatus (Ochlerotatus) vigilax , the common saltmarsh mosquito. In March 1996, eggshells were buried in soil of five mosquito-breeding sites on Kooragang Island, NSW. Eggshells were graded by colour using Munsell^® soil-colour charts. Seventy-five percent of newly hatched eggshells were black and 25% were dark brown in colour. Black eggshells faded quickly to lighter shades of brown, so that none were recovered 20 months after commencement of the study. Eggshells faded to a median of dark brown after 13 months and medium brown after 2 years. In another study, significant differences in colour frequency between existing saltmarsh and two recently created oviposition sites were found; the latter site being characterised by numerous black and dark-brown eggshells and very few light-brown eggshells. The results suggest that active and inactive oviposition sites may be identified using the frequency distribution of eggshell colours based on large numbers of eggshells, whereas the colour of individual eggshells only coarsely reflects age since eclosion.