Axenic Larvae Research Articles

Mosquitoes rely on their gut microbiota for development.

Field studies indicate adult mosquitoes (Culicidae) host low diversity communities of bacteria that vary greatly among individuals and species. In contrast, it remains unclear how adult mosquitoes acquire their microbiome, what influences community structure, and whether the microbiome is important for survival. Here, we used pyrosequencing of 16S rRNA to characterize the bacterial communities of three mosquito species reared under identical conditions. Two of these species, Aedes aegypti and Anopheles gambiae, are anautogenous and must blood-feed to produce eggs, while one, Georgecraigius atropalpus, is autogenous and produces eggs without blood feeding. Each mosquito species contained a low diversity community comprised primarily of aerobic bacteria acquired from the aquatic habitat in which larvae developed. Our results suggested that the communities in Ae. aegypti and An. gambiae larvae share more similarities with one another than with G. atropalpus. Studies with Ae. aegypti also strongly suggested that adults transstadially acquired several members of the larval bacterial community, but only four genera of bacteria present in blood fed females were detected on eggs. Functional assays showed that axenic larvae of each species failed to develop beyond the first instar. Experiments with Ae. aegypti indicated several members of the microbial community and Escherichia coli successfully colonized axenic larvae and rescued development. Overall, our results provide new insights about the acquisition and structure of bacterial communities in mosquitoes. They also indicate that three mosquito species spanning the breadth of the Culicidae depend on their gut microbiome for development.

Social attraction mediated by fruit flies' microbiome.

Larval and adult fruit flies are attracted to volatiles emanating from food substrates that have been occupied by larvae. We tested whether such volatiles are emitted by the larval gut bacteria by conducting tests under bacteria-free (axenic) conditions. We also tested attraction to two bacteria species, Lactobacillus brevis, which we cultured from larvae in our lab, and L. plantarum, a common constituent of fruit flies' microbiome in other laboratory populations and in wild fruit flies. Neither larvae nor adults showed attraction to axenic food that had been occupied by axenic larvae, but both showed the previously reported attraction to standard food that had been occupied by larvae with an intact microbiome. Larvae also showed significant attraction to volatiles from axenic food and larvae to which we added only either L. brevis or L. plantarum, and volatiles from L. brevis reared on its optimal growth medium. Controlled learning experiments indicated that larvae experienced with both standard and axenic used food do not perceive either as superior, while focal larvae experienced with simulated used food, which contains burrows, perceive it as superior to unused food. Our results suggest that flies rely on microbiome-derived volatiles for long-distance attraction to suitable food patches. Under natural settings, fruits often contain harmful fungi and bacteria, and both L. brevis and L. plantarum produce compounds that suppress the growth of some antagonistic fungi and bacteria. The larval microbiome volatiles may therefore lead prospective fruit flies towards substrates with a hospitable microbial environment.

Significance of bacteria in oviposition and larval development of the sand fly Lutzomyia longipalpis

BackgroundMicrobial ecology of phlebotomine sand flies is not well understood although bacteria likely play an important role in the sand fly biology and vector capacity for Leishmania parasites. In this study, we assessed the significance of the microbial community of rabbit feces in oviposition and larval development of Lutzomyia longipalpis as well as bacterial colonization of the gut of freshly emerged flies.MethodsSterile (by autoclaving) and non-sterile (control) rabbit feces were used in the two-choice assay to determine their oviposition attractiveness to sand fly females. Bacteria were identified by amplification and sequencing of the 16S rRNA gene with universal eubacterial primers. Sterile, control (non-sterile), and sterilized and inoculated rabbit feces were used to assess the significance of bacteria in L. longipalpis development. Newly emerged adult flies were surface-sterilized and screened for the bacterial population size and diversity by the culturing approach. The digestive tract of L4 sterile and control larvae was incubated with Phalloidin to visualize muscle tissues and DAPI to visualize nuclei.ResultsTwo-choice behavioural assays revealed a great preference of L. longipalpis to lay eggs on rabbit feces with an active complex bacterial community (control) (85.8 % of eggs) in comparison to that of sterile (autoclaved) rabbit feces (14.2 %). Bioassays demonstrated that L. longipalpis larvae can develop in sterile rabbit feces although development time to adult stage was greatly extended (47 days) and survival of larvae was significantly lower (77.8 %) compared to that of larvae developing in the control rabbit feces (32 days and 91.7 %). Larval survival on sterilized rabbit feces inoculated with the individual bacterial isolates originating from this substrate varied greatly depending on a bacterial strain. Rhizobium radiobacter supported larval development to adult stage into the greatest extent (39 days, 88.0 %) in contrast to that of Bacillus spp. (76 days, 36.0 %). From the complex natural bacterial community of rabbit feces, R. radiobacter survived pupation and colonized the newly emerged females most successfully (82.6 % of all bacteria cultured); however, only 25 % of females were positive for bacteria in the digestive tract upon emergence. Immunohistochemistry did not reveal any obvious differences in anatomy of the digestive tract between control and axenic larvae.ConclusionsThe bacterial community in the sand fly larval habitat affects oviposition and larval development although bacteria are not essential for successful development of L. longipalpis. Different bacteria contribute to larval development to various degrees and some, e.g. Rhizobium radiobacter, survive pupation and colonize the digestive tract of newly emerged females. With the establishment of the axenic rearing system, this study opens new venues to study the effect of bacteria on the gut epithelial immunity and vector competence of sand flies for Leishmania parasites with a goal to develop paratransgenic approaches for Leishmania control.

Two strategies to unravel gene expression responses of host-microbe interactions in cod (Gadus morhua) larvae

The commensal bacteria in the intestine play essential roles in the development and functionality of the host. To unravel the host–microbe interactions in Atlantic cod (Gadus morhua L.) larvae, we used two molecular approaches: (1) suppression subtractive hybridization-polymerase chain reaction (SSH-PCR) to identify host gene responses and (2) expression analysis of selected genes reported to be differentially expressed in gnotobiotic zebrafish in a previous study to determine whether these host responses are also conserved in cod. Suppression subtractive hybridization-PCR identified 156 transcripts putatively regulated by the presence of bacteria. However, out of 22 selected transcripts, only four were significantly differentially expressed when quantified using quantitative (real-time) PCR. Expression analysis of selected genes from zebrafish revealed possible conservation of host responses for three out of eight genes analysed. For most of the genes quantified, the gene expression pattern varied between two biological replicates. This may reflect differences in the bacterial composition in the rearing bottles, and denaturing gradient gel electrophoresis analysis confirmed significant differences between the two replicates with regard to bacterial diversity. The varying effects on gene expression caused by differences in the microbial composition show the necessity of further studies where axenic cod larvae are compared with larvae raised in defined and controlled (gnotobiotic) environments.

A method to obtain axenic Angiostrongylus vasorum first-stage larvae from dog feces.

An improved method to obtain a large number of axenic larvae of Angiostrongylus vasorum from fecal samples was developed in the present study. The procedure here in reported consisted of obtaining larvae using a modified Baermann technique, followed by an additional filtration step. This isolation technique recovered almost 90% of the living larvae in a clean preparation. Isolated larvae were submitted to decontamination treatments with either sodium hypochlorite or antibiotic cocktail solutions. The axenic status, as confirmed by oral inoculation of decontaminated larvae into germ-free mice, was only achieved using larvae treated with 0.5% sodium hypochlorite solution for 10 min. The isolation and decontamination treatment did not affect larval viability. Treated larvae remained viable and infective to the invertebrate host.

Experimental herpes-like viral infections in marine bivalves: demonstration of interspecies transmission.

Since 1972, herpes-like virus infections have been reported in several marine bivalve species around the world. Viral detection was often associated with high mortality rates in larvae and spat. To determine whether a single virus is able to infect different bivalve host species, we carried out experimental transmission assays. As a first step, 8 assays were performed to infect axenic Crassostrea gigas larvae with virus from infected C. gigas larvae using a previously described protocol. The protocol appeared reliable and PCR was confirmed as a powerful technique for detecting viral DNA in experimentally infected oysters. The defined protocol was then applied to infect different bivalve species. Interspecies viral transmission was demonstrated under laboratory conditions. The same phenomenon may occur in private hatcheries and may be promoted by intensive rearing conditions. This hypothesis is reinforced by reports of concomitant mortalities in the larvae of several bivalve species and by the first molecular analysis of infected larval samples.

A method of decontaminating Strongyloides venezuelensis larvae for the study of strongyloidiasis in germ-free and conventional mice

To study the possible influence of intestinal micro-organisms on the course of strongyloidiasis in mice, a method was developed to obtain axenic infective larvae of Strongyloides venezuelensis. Cultured larvae from conventional mice were treated with sodium hypochlorite 0.25% for 10 min, washed in distilled water and then exposed to various combinations of antibiotics for 30 or 60 min. Success was achieved with a combination of penicillin 180 mg/L and ceftazidime 1 mg/ml. Decontamination of the larvae was determined by aerobic and anaerobic culture and by inoculation into gnotobiotic mice. Viability was established by subcutaneous inoculation of larvae into germ-free and conventional mice. Preliminary results showed that gnotobiotic mice were more susceptible than conventional mice to infection with axenic S. venezuelensis larvae as judged by faecal egg excretion, recovery of worms in the small intestine and histopathological examination of the duodenal mucosa. These results suggest that the normal intestinal flora protects the host against experimental infection with S. venezuelensis.

Influence of Microorganisms on Alfalfa Leafcutter Bee ( Megachile rotundata) Larval Development and Susceptibility to Ascosphaera aggregata

The influence of microorganisms associated with alfalfa leafcutter bee ( Megachile rotundata) larvae on chalkbrood caused by Ascosphaera aggregata was determined by comparing disease in agnotobiotic and axenic larvae, with and without an introduced microflora (gnotobiotic). Bacterial and fungal populations in the frass of larvae reared on natural provisions (agnotobiotic) were log 4.24 and log 3.90 cfu/g of frass, respectively. No fungi were recovered from the frass of larvae reared on γ-irradiated provisions, but bacterial populations averaged log 0.89 cfu/g (near-axenic). Following ingestion of ascospores of A. aggregata, the incidence of chalkbrood was significantly higher in agnotobiotic (67.6%) than near-axenic (50.2%) larvae, but the time to death of infected larvae was the same for both. Microorganisms were inoculated onto the surface of γ-irradiated provisions adjacent to leafcutter bee eggs and were ingested by larvae; 11 of 13 bacteria, 9 of 10 filamentous fungi, but 0 of the 7 yeasts were recovered from frass (gnotobiotic). Populations of inoculated bacteria and filamentous fungi recovered in frass ranged from log 5.40 to 7.30 cfu/g and log 5.21 to 6.52 cfu/g of frass, respectively. Of the 20 microorganisms recovered in frass, only Bacillus brevis and a Streptomyces sp. significantly increased the incidence of chalkbrood (74.0 and 74.8%, respectively) when larvae were coinoculated with ascospores of A. aggregata (1:1 ratio); the incidence of chalkbrood in larvae inoculated with the pathogen alone was 52.1%. None of the microorganisms themselves appeared to be deleterious to larvae. There were no significant differences in microbial populations in frass from healthy larvae or chalkbrood cadavers; however, noninoculated bacteria were associated with all nine fungal treatments. The results of this study suggest that microorganisms associated with the alimentary tracts of alfalfa leafcutter bee larvae have a minimal impact if any on chalkbrood.

Bacterivory in Pacific oyster Crassostrea gigas larvae

A bacterium (Strain CA2) that enhances sunrival and growth of larvae of the oyster Crassostrea gigas (Thunberg) was used in a series of experiments to determine the occurrence of bacterivory in straight-hinged bivalve larvae. Size and carbon content of this bacterium was found to be within the range reported for naturally occurnng marine bacteria. Unattached, motile, DAPI-stained CA2 cells were readily captured and ingested by oyster larvae and were seen to accumulate in larval digestive systems. Ingestion of 14C-labelled bacteria occurred at all bacterial concentrations tested from 1 X 105 to 1 X 10' cells m]-'. Retention of carbon by axenic oyster larvae, fed either 14C-labelled live or heat-killed bacteria in 'pulse-chase' feeding experiments, demonstrates the endogenous ability of larvae to digest and assirmlate bacterial carbon.

Experimental manipulations of the organic compositions of seawater: implications for studies of energy budgets in marine invertebrate larvae

Correct measurement of changes in biomass and metabolic rates over time are two essential elements for the accurate construction of energy budgets for invertebrate larvae. Both components of larval energetics are altered by changes in the organic chemistry of the seawater. Axenic (bacteria-free) veliger larvae (88 μm shell length) of the bivalve Crassostrea gigas (Thunberg, 1795) had a 53% enhancement of their metabolic rate relative to control values (5.8 ± 0.6 pmol larva −1 h −1, x ± 1 se ) when exposed to seawater to which 1 μM glucose had been added. Gastrulae, prism-stage, and pluteus-stage, larvae of the sea urchin Lytechinus pictus (Verrill, 1867) had significantly higher metabolic rates when 1 μM amino acids (16 amino acids at 62.5 nM each) was added to seawater. Gastrulae increased their rate of respiration by 35%, from 10 ± 0.9 to 13.5 ± 1.4 pmol O 2 embryo −1 h −; prism-stage larvae by 33% from 40.9 ± 2.0 to 54.4 ± 2.8; and pluteus-stage larvae by 50%, from 33.4 ± 1.5 to 50.3 ± 3.1. Lecithotrophic larvaei of the gastropod Haliotis rufescens Swainson, 1822, either had no change (Day 1, trochophore larvae) or a significant increase (Day 2, veliger larvae) in dry organic weight when reared in natural seawater that had been passed through a filter of 0.2-μm pore size (to remove particles). In contrast, sibling larvae always decreased in dry organic weight when reared in seawater which had first been passed through a sand-filter (a treatment that alters the organic chemistry of seawater), and then a 0.2-μm (pore size) filter. These data show that alterations of the organic chemistry of seawater can affect the growth and metabolism of invertebrate larvae. If such modifications are not controlled, energy budgets constructed from laboratory experiments on larvae in altered seawater may bear little resemblance to the energetics of larvae in the field.

Infection byBeauveria bassiana ofLeptinotarsa decemlineata larvae as a consequence of fecal contamination of the integument followingper os inoculation

A study ofper os inoculated larvae was undertaken to determine if the entomopathogenic fungus,Beauveria bassiana, is able to infect the Colorado potato beetle,Leptinotarsa decemlineata, via the alimentary tract. Surface orper os inoculated larvae which were immediately surface sterilized post inoculation did not succumb to infection, whereas those larvae not sterilized became infected. Histological studies of fed or starved, agnotobiotic (with microbial flora) and axenic larvae revealed that conidia can germinate in the gut regardless of the presence of gut microflora. However, infection via the alimentary tract was never observed in fed larvae and only noted in a single starved individual. It is concluded that infections ofper os inoclated larvae occurred after surface contamination of the integument by viable conidia contained in the frass. The rate of food passage through the gut is probably important in preventingper os infections.

Amino acid fluxes to and from seawater in axenic veliger larvae of the bivalve (Crassostrea gigas)

Rates of uptake of dissolved amino acids were determined for axenic veliger larvae of the bivalve Crassostrea gigas (70 km shell length). Analyses of net fluxes from seawater of amino acids by high-performance liquid chromatography showed a net uptake of all acidic, basic, and neutral amino acids tested. From a substrate concentration of 100 n M each, valine had the hlghest rate of uptake at 70 fmol larva-' h-', and arginine the lowest at 4 fmol larva-' h-' The influx of alanine, arginine, glutamic acid and leucine (measured radiochemically) reflected the net flux (measured by chromatography). However, there was a net efflux of taurine (0.58 pm01 larva-' h-') even though isotope experiments revealed an influx of 0.32 pm01 larva-' h-' Efflux of taurine was also observed for nonaxenic veligers. Axenic veligers had a K, (substrate concentration at half J,,,) of 11.4 pM and a J,,, (maximum transport capacity) of 4.6 pm01 larva-' h-' for alanine; for leucine, the corresponding values were 7.1 pM and 2.2 pm01 larva-' h-' At substrate concentrations near the K, values (on the order of 10 l t M ) , C gigas larvae could account for 100 % of their oxidative needs by the uptake of amino acids from seawater In most marine environments, such high concentrations are limited to seawater near the sediment. Thus, the overall contribution of dissolved amino acids will depend on the substrate concentration encountered by the larvae in nature.

The origin of sheep blowfly,Lucilia cuprina(Wiedemann) (Diptera: Calliphoridae), attractants in media infested with larvae

AbstractLaboratory bioassays with gravid females ofLucilia cuprina(Wiedemann) were used to isolate the source(s) of olfactory attractants emanating from larvae-infested media. Adults were not attracted by odours from axenic (micro-organism-free) larvae, but volatiles from xenic larvae were highly attractive. The attractants proved to be kairomones not pheromones, as odours from other species of calliphorids and a sarcophagid species were also attractive. Axenic, proteinaceous media produced a low level of attractive volatiles, which was increased by the activities of axenic larvae growing on the media. A greater degree of attraction occurred to odours from xenic media, and this too was much increased by the actions of growing larvae. The order of attractiveness of such volatiles is therefore: xenic with larvae >> xenic without larvae > axenic with larvae > axenic without larvae. It is concluded that larvae-infested media owe their great attractiveness to the volatiles produced by the action of micro-organisms, not to specific larval volatiles. Larval activity accentuates the output of attractive volatiles from both xenic and axenic proteinaceous media, possibly due to the effects of digestive enzymes, pH changes, mechanical mixing, warming or aeration or a combination of some or all of these factors.

Requirement of exogenous inducers for metamorphosis of axenic larvae and buds of Cassiopea andromeda (Cnidaria: Scyphozoa)

Planula larvae and asexually-produced buds of the rhizostome scyphozoan Cassiopea andromeda (collected throughout the year in Eilat, Israel) have the ability, under axenic conditions, to attach to a substrate and undergo morphogenetic development to form a polyp (=scyphistoma) in: (1) the presence of unidentified inducers found in the adult habitat and (2) the presence of cefined organic compounds. Axenic planulae and buds were unable to settle and complete metamorphosis in autoclaved artificial or natural seawater from the North Sea when maintained without food, but continued swimming while decreasing in size and protein content, eventually dying within three months. When maintained in autoclaved seawater from the Red Sea, between 25 and 46% of the planulae and 4 and 11% of the buds metamorphosed within 30 d. Axenic solutions of cholera toxin, thyroid stimulating hormone, and pancreatic casein hydrolysate peptides in artificial seawater induced morphogenic development of 20 to 100% of planulae and buds within 2 to 18 d. The natural inducer(s) in Red Sea seawater, though unidentified, may have characteristics similar to the large proteins and small peptide inducers used in this study. Planulae and buds older than 20 d metamorphosed sooner and responded to lower concentrations of pancreatic casein hydrolysate peptides than younger individuals. This may be a physiological mechanism for enhancing metamorphosis and survival in nature. The data show that settlement and metamorphosis can be induced by solutions of cholera toxin and thyroid stimulating hormone, suggesting that, as in mammalian systems, the mechanism of action of these chemicals may involve cyclic adenosine monophosphate (cAMP) as an intermediate messenger. However, dibutyric cAMP, which is capable of passing through membranes and functioning normally inside the cell, did not induce metamorphosis of buds, and the levels of intracellular cAMP in buds and larvae typically increased slowly during induction of metamorphosis, unlike the high and rapid increases associated with cAMP-mediated biochemical events in mammalian cells. These results suggest that the observed cAMP changes seen were associated with metamorphic development, but not with the triggering mechanism.

Increased activity of blood lysozyme in the greater wax moth larvae affected perorally by bacterial invaders with chitinase added

1. 1. Larvae of the greater wax moth induced perorally with entomopathogenic bacterial invaders with chitinase (EC 3.2.1.14) added, increase the concentration of lysozyme in their haemolymphs. The immune defence system in insects infected orally by the parasite contaminated diet without chitinase is however not affected. 2. 2. By altering the protective chitinous barriers in the intestines, chitinase enhances the action of bacterial parasites used as inducers of insect immune response. 3. 3. Although orally induced larvae with Serratia marcescens, Pseudomonas aeruginosa, Bacillus thuringiensis var. kurstaki or Xenorhabdus nematophilus detectably elevate the lysozyme concentration, they do not withstand the challenge and die with a typical sign of Pseudomonas aeruginosa bacteriemia. The increase of lysozyme syntheis but lack of protective immunity against P. aeruginosa provides the evidence that not all components of the insect defence system are induced in perorally stimulated larvae of the moth. 4. 4. An indigenous bacterial flora of the greater wax moth intestines acts additionally as an inducer of immune response in chitinase treated larvae since the increase of lysozyme activity in monoxenic insects is higher than it is in axenic larvae devoid of S. faecium.

Efforts to induce defence responses in the greater wax moth larvae by oral feeding of insect pathogenic bacteria

1. 1. Attempts were made to stimulate or induce the internal defence system in the greater wax moth larvae by oral feeding of the insect non-sporulating bacterial parasites Pseudomonas aeruginosa, Serratia marcescens or Xenorhabdus nematophilus. 2. 2. After ingesting pathogens, an acquired immunity against an intrahaemocoelic challenge dose of viable P. aeruginosa cells was not found in the three insect model systems: monoxenic, axenic and germ-free larvae, although in some treatments a negligibly elevated level of blood lysozyme activity appeared. 3. 3. Animals given an injection of foreign soluble or particulate molecules directly into the larval haemocoel, including saline W, a physiological salt solution for lepidopterous larvae, showed an apparently increased concentration of blood lysozyme and withstood challenge with a lethal dose of P. aeruginosa. 4. 4. These results are discussed in terms of the consideration that chitinase activity may affect the insect gut barriers enabling the contact of bacterial invaders with epithelial gut cells, and therefore it may help to demonstrate the inducible immunity by the oral route in a more convenient model system.

Na+-dependent amino acid transport in bacteria-free sea urchin larvae

1. Influx of14C-labeled serine and leucine into axenic larvae ofStrongylocentrotus purpuratus occurs from substrate concentrations as low as 125 nmolar. The relation between substrate concentration and the rate of influx has the form of a rectangular hyperbola and is well described by the Michaelis-Menten equation. The apparent kinetic constantsJi max andKt are both dependent on the sodium concentration of the medium. 2. Analysis of the relation between the measured influx of serine or leucine, and sodium concentration suggests that two sodium ions are transported per amino acid. The data also suggest cooperative interaction between the binding sites for both sodium and the amino acid substrate. 3. We provide the first clear evidence of sodium coupled net entry of 9 neutral amino acids, as determined by high performance liquid chromatography. We propose a model for transport of the form (Na+)2·(carrier)·(S) and provide evidence that this quaternary complex is translocated intact across the cell surface.

GROWTH STUDIES WITH BACTERIA-FREE OYSTER (CRASSOSTREA GIGAS) LARVAE FED ON SEMI-DEFINED ARTIFICIAL DIETS

By the aseptic removal of gametes and in vitro fertilization of Crassostrea gigas eggs it was possible to obtain axenic larvae without the use of antibiotics. Optimal culture conditions for maintaining axenic larvae on algal diets have been developed.Based on the results of ten growth experiments, C. gigas larvae fed on a semidefined biphasic artificial diet had a mean shell length of 96.0 µm (standard deviation ± 3.0 µm) after 6 days of growth. This was significantly greater (P < 0.001) than the mean shell length of starved larvae (79.8 ± 0.8 µm), but was significantly less (P < 0.001) than that of larvae fed on the alga Chaetoceros calitrans (113.6 ± 8.0 µm). C. gigas larvae grew on the dissolved organic fraction of the artificial diet alone, but growth was less than with the complete biphasic diet. This is the first reported demonstration that bivalve larvae can utilize dissolved nutrients for growth under axenic conditions where the possible nutritional contribution of bacteria is completely eliminated.

Studies on the Nutritional Relationship of LarvalAedes Aegypti(Diptera: Culicidae) withSmittium Culisetae(Trichomycetes)

SUMMARY Axenic larvae of the mosquito, Aedes aegypti, were reared singly on a sterile semidefined medium from which required vitamins and sterols were individually deleted. Trichospores obtained from axenic cultures of the fungus, Smittium culisetae (Trichomycetes), were added to the medium to infest the hindguts of the larvae. First instar larvae invariably became heavily infested from the original spore inoculum, but infestations were often not maintained in the subsequent larval instars. In some instances thalli attached to the external cuticle of living larvae. With the introduction of S. culisetae, larvae were able to attain at least one additional instar when reared without riboflavin, pyridoxine or nicotinamide. No such improvement in larval growth was noted with S. culisetae upon the omission of thiamine or Ca pantothenate. In the absence of sterols larvae did not develop beyond the fourth instar, but following the addition of S. culisetae, one out of 45 larvae pupated. Both the lipid extract and dead whole mycelium of S. culisetae are adequate sources of sterols for larvae, and larvae are able to utilize desmosterol, the predominant sterol in Smittium spp. Sparse free mycelial growth was evident in the medium in those vitamin and sterol tests where larvae demonstrated an improvement in growth; hence the free mycelium may have been serving as a dietary source of nutrients. It is possible that mosquito larvae growing under some conditions of nutritional stress may be provided with required nutrients through infestations of S. culisetae in their guts.

Mode of formulation of cholesta-5,7-dien-3beta-ol from Cholest-5-en-3beta-ol by Larvae of Calliphora erythrocephala.

1. The conversion of cholest-5-en-3beta-ol (cholesterol) into cholesta-5,7-dien-3beta-ol by axenic Calliphora erythrocephala larvae was demonstrated. 2. The transformation is probably direct (Delta(5)-->Delta(5,7)) and does not involve a Delta(0) intermediate (Delta(5)-->Delta(0)-->Delta(7)--> Delta(5,7)). 3. Delta(7)-bond formation involves the stereospecific elimination of the 7beta hydrogen atom. 4. The relative amounts of free and esterified sterols were determined in larvae grown on cholesterol as sole sterol source and on 5alpha-cholestan-3beta-ol supplemented with minimal amounts of cholesterol. 5. The significance of the results is assessed in relation to the probable role of cholesta-5,7-dien-3beta-ol as an intermediate in the biosynthesis of ecdysones.