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Ferritin is a ubiquitous multi-subunit iron storage protein, made up of heavy chain and light chain subunits. In recent years, invertebrate ferritins have emerged as an important, yet largely underappreciated, component of host defense and antioxidant system. Here, two alternatively spliced transcripts encoding for a unique ferritin heavy chain homolog (MdFerH), and a transcript encoding for a light chain homolog (MdFerL) are cloned and characterized from Musca domestica. Comparing with MdFerH1, a fragment is absent at the 5' untranslated region of MdFerH2, where a putative iron response element is present. Amino acid sequence analysis shows that MdFerH possesses a strictly conserved ferroxidase site, while MdFerL has a putative atypical active center. Tissue distribution analysis indicates that MdFers are enriched expressed in gut. When the larvae receive diverse stimulations, including challenge by bacteria, exposure to excess Fe2+, doxorubicin or ultraviolet, the expression of MdFers is positively up-regulated in different degrees and different temporal patterns, indicating their potential roles in oxidative stress. The two mRNA isoforms of MdFerH appear to be differentially expressed in different tissues, but seem to show the similar expression patterns under diverse stress conditions. Further investigation reveals that silencing MdFers can alter the redox homeostasis, leading elevated mortalities of larvae following bacterial infection. Inspiringly, recombinant MdFerL produced in Pichia pastoris shows significant iron-chelating activity in vitro. These results suggest a pivotal role of ferritins from housefly in iron homeostasis, antibacterial immunity and redox balance.

Until now there is no molecular model of starch digestion and absorption of the resulting glucose molecules along the larval midgut of Musca domestica. For addressing to this, we used RNA-seq analyses from seven sections of the midgut and carcass to evaluate the expression level of the genes coding for amylases, maltases and sugar transporters (SP). An amylase related protein (Amyrel) and two amylase sequences, one soluble and one with a predicted GPI-anchor, were identified. Three highly expressed maltase genes were correlated with biochemically characterized maltases: one soluble, other glycocalyx-associated, and another membrane-bound. SPs were checked as being apical or basal by proteomics of microvillar preparations and those up-regulated by starch were identified by real time PCR. From the 9 SP sequences with high expression in midgut, two are putative sugar sensors (MdSP4 and MdSP5), one is probably a trehalose transporter (MdSP8), whereas MdSP1-3, MdSP6, and MdSP9 are supposed to transport glucose into cells, and MdSP7 from cells to hemolymph. MdSP1, MdSP7, and MdSP9 are up-regulated by starch. Based on the data, starch is at first digested by amylase and maltases at anterior midgut, with the resulting glucose units absorbed at middle midgut. At this region, low pH, lysozyme, and cathepsin D open the ingested bacteria and fungi cells, freeing sugars and glycogen. This and the remaining dietary starch are digested by amylase and maltases at the end of middle midgut and up to the middle part of the posterior midgut, with resulting sugars being absorbed along the posterior midgut.

It is now widely accepted that Hexapoda emerged from Crustacea. Compared to the ubiquitous calcified exoskeleton in crustaceans, a mineralized cuticle in insects is extremely rare. Catecholamine-driven protein cross-links play a leading role in the sclerotization of insect cuticle. In this study, mineralization was discovered in the pupal cuticle of Bactrocera dorsalis (Diptera: Tephritidae), a common pest of fruit farms. We mainly profiled the features of mineralized pupal cuticles from B. dorsalis and its white mutant B. dorsaliswh and unmineralized cuticle from Musca domestica using high-resolution field emission scanning electron microscopy (SEM) and transmission electron microscopy (TEM) combined with structural analysis involving infrared (IR) spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and synchrotron X-ray absorption near-edge structure (XANES) spectroscopy. We also compared the thermodynamic and mechanical properties of different pupal cuticles. The results showed that the pupal cuticles of B. dorsalis contain a phase of stable amorphous calcium phosphate (ACP) with a high level of magnesium, which is mainly distributed in the exocuticle and assists in the formation of a graded, stiffened cuticle structure. Unexpectedly, this ACP possesses a very low Ca/P ratio and has a composition similar to that of CaHPO4·2H2O. The degree of mineralization in the pupal cuticle of B. dorsaliswh (approximately 22 wt%) is significantly greater than that of wild-type B. dorsalis (approximately 12 wt%), which indicates that there may be a connection between the biomineralization and tyrosine-mediated tanning pathways. These findings provide new evidence for the mineralization of the insect cuticle, which may shed new light on the evolutionary mechanism underlying the divergence of cuticle sclerotization between insects and crustaceans.

l-Glutamic acid (glutamate) mediates fast inhibitory neurotransmission by affecting glutamate-gated chloride channels (GluCls) in invertebrates. The molecular function and pharmacological properties of GluCls have been well studied, but not much is known about their physiological role and localization in the insect body. The distribution of GluCls in the housefly (Musca domestica L.) was thus compared with the distribution of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls). Quantitative PCR and ligand-binding experiments indicate that the GluCl and GABACl transcripts and proteins are predominantly expressed in the adult head. Intense GluCl immunostaining was detected in the lamina, leg motor neurons, and legs of adult houseflies. The GABACl (Rdl) immunostaining was more widely distributed, and was found in the medulla, lobula, lobula plate, mushroom body, antennal lobe, and ellipsoid body. The present findings suggest that GluCls have physiological roles in different tissues than GABACls.

As important molecular chaperones, members of the 70kDa heat shock protein (HSP70) family play essential roles in stress tolerance and innate immunity in organisms. The full-length complementary DNA (cDNA) of a novel inducible HSP70, named as MdHSP70, was isolated from Musca domestica. The cDNA clone consisted of 2411 bp with a 1956 bp open reading frame which encodes 651 amino acids. Using real-time quantitative polymerase chain reaction (qPCR), we investigated the transcriptional profile of the gene under heat shock, cadmium stress and in response to bacteria. Increased expression of MdHSP70 was observed in response to both heat shock and Cd stress. The expression of MdHSP70 was significantly induced by Escherichia coli or Staphylococcus aureus stimulation. Larvae were fed bacteria expressing dsRNA targeting the MdHSP70 gene. Our results showed high mortality in larvae treated with dsRNA of MdHSP70 at heat shock, Cd stress and bacterial invasion, suggesting that MdHSP70 is potentially involved in the stress and immune responses of the house fly and perhaps contributes to protection against cellular injury.

Water loss from adult male houseflies was continuously recorded using a flow-through humidity meter, which enabled losses to be apportioned between the sum of cuticular and respiratory transpiration, salivation and excretion. Transpiration accounted for >95% of water lost from sham-injected flies, compared with excretion (3.0%) and salivation (2.4%). In contrast, excretion accounted for 40% of water lost from flies injected with > or =3 microl of saline, whereas salivary losses were unchanged. Saline injections (1-5 microl) expanded the abdomen in the dorsal-ventral plane, and this expansion was positively correlated with the magnitude of the ensuing diuresis, suggesting the signal for diuretic hormone release originates from stretch receptors in abdominal tergal-sternal muscles. The effects of decapitation, severing the ventral nerve cord within the neck or ligaturing the neck, showed the head was needed to initiate and maintain diuresis, but was neither the source of diuretic hormone nor did it control the discharge of urine from the anus. These findings indicate the head is part of the neural-endocrine pathway between abdominal stretch receptors and sites for diuretic hormone release from the thoracic-abdominal ganglion mass. Evidence is presented for Musdo-K having a hormonal role in the control of diuresis, although other neuropeptides may also be implicated.

Programmed cell death (PCD) and phagocytotic activity of immune cells play a pivotal role in insect development. We examined the influence of Zn 2+ , an important element to fundamental biological processes, on phagocytosis and apoptosis of hemocytes in two fly species: Musca domestica and Drosophila melanogaster. Hemocytes were isolated from the third instar larvae of both species and treated for 3 h with zinc chloride solutions, containing 0.35 mM or 1.7 mM of Zn 2+ , and untreated as control. Phagocytotic activity of hemocytes was examined by flow cytometry after adding latex fluorescent beads to the medium, while apoptosis was evaluated by application of annexinV–FITC and pan-caspase-FITC inhibitor. Mitochondrial viability was determined by measuring resazurin absorbancy in the cell medium. The obtained results showed that Zn 2+ increases phagocytosis and affects PCD of both species hemocytes but each in a different way. Zinc decreases fraction of annexin-positive hemocytes in M. domestica but increases it in D. melanogaster . The pan-caspase analysis revealed low and high activity of caspases in hemocytes of M. domestica and D. melanogaster , respectively. Zn 2+ also decreased the viability of hemocyte mitochondria but only in D. melanogaster . It suggests that flies use different pathways of PCD, or that Zn plays a different role in this process in M. domestica than in D. melanogaster .

The staining intensity (median neurosecretory cell index) of the median neurosecretory cells (MNC) in Musca domestica increased as oogenesis progressed from stages 2 to 10. The amount of neurosecretory material within the MNC was dependent upon the presence of ovaries with developing or mature follicles. Ovariectomized flies had a median neurosecretory index that was 50 per cent less than that of control flies with mature eggs. In addition, we found that ring gland removal decreased the staining frequency of three different neurosecretory cell groups; increased staining frequency in another; increased the amount of neurosecretory material within the MNC fibre tract; increased the cytoplasmic area of types A and A′ MNC. Furthermore, neither the juvenile hormone analogue nor the ring gland had a direct effect on the median neurosecretory cell index but did influence neurosecretory activity indirectly by activating the ovaries. We hypothesize that an ovarian hormone—the oostatic hormone—regulates either the release from or synthesis of neurosecretory material within the MNC.

The distribution of choline metabolites has been studied in the nervous system of housefly larvae reared on diets containing various amounts of choline or on diets containing either β-methylcholine (β-MCh) or N-dimethylethylcholine (DMECh). Adult houseflies were obtained from larvae reared on diets containing at least 4·6 times the level of choline in the basic diet or on diets containing the two choline analogues. The distribution of choline metabolites has been studied in the heads and thoraces plus abdomina of these adults. Acetylcholine was found to be concentrated in the larval nervous system where it was synthesized preferentially to other choline metabolites when the amount of choline available to the insect was severely restricted. The acetylcholine content of the adult was between six and nine times that of the larval nervous system and approximately 70 per cent was concentrated in the head. The amount of acetylcholine present in flies obtained from larvae fed on diets containing the lowest amount of choline which allowed adults to develop was 230 pmole/insect. Flies obtained from larvae fed on diets with added β-MCh contained 150 pmole of acetylcholine/insect but no detectable acetyl-βMCh. Flies obtained from larvae fed on diets with added DMECh contained 43 pmole of acetylcholine and 127 pmole of acetyl-DMECh/insect. It is concluded that the two choline analogues spare the choline requirement of the housefly by two different mechanisms. β-MCh displaces choline from the lipids of non-nervous tissue making more available for uptake into the nervous system where sufficient acetylcholine is synthesized for adult development. DMECh is a complete replacement for choline; the acetyl-DMECh acting as a neurotransmitter in place of acetylcholine.

Changes in the giant mitochondria (sarcosomes) in the flight muscles of the ageing housefly Musca domestica L. have been quantitatively determined and the results incorporated in the present paper. Variations in mitochondrial size and number are both sex-related and age-dependent. In the male fly both mean size as well as the total number of mitochondria per fly increase up to the beginning of the second week and then begin to decline by the end of the second week. In the female, the maximum size and number are also reached by the third day and remain unchanged until the end of the third week, at which time both size and number then show a dramatic decline. From observations of specific changes in the populations of differently sized sarcosomes, it is inferred that multiplication, growth, and lysis of mitochondria are continuous processes throughout the life-span of the flies, but that their rates differ with age. In the male fly these changes in size and number of the mitochondria are preceded by the loss of wings and a decline in ATP-ase and in alpha-glycerophosphate dehydrogenase activities, whereas in the female fly such mitochondrial variations occur with the decline in the physical and biochemical functions of flight.

Bioinduction of internal melanotic tumours and melanotic sclerosis in adult male and female houseflies was accomplished by elevating the l -phenylalanine content of a fully chemically defined diet beyond 1 per cent levels. A high frequency of tumour formation and sclerosis was dependent on the presence of eight essential l -amino acids in the diet: tryptophan, phenylalanine, leucine, isoleucine, valine, arginine, lysine, and threonine. Methionine was non-essential for tumour formation in males and intermediate in its position between essential and non-essential acids for female tumour formation. l -Histidine, essential for ovarian development, was non-essential for tumour formation. Tumour formation and sclerosis appeared to be related directly to the animal's ability to synthesize protein, presumably in the form of enzymes or other proteins required for melanin formation. Supplementing the tumour-inducing (TuI) diet with RNA (0·4%) was somewhat effective in reducing tumour frequency whereas DNA was without effect. Individual purine or pyrimidine bases reduced tumour frequency by as much as 90 per cent of controls. Ribose and deoxyribose nucleosides and nucleotides were without significant effect with two important exceptions. Guanosine and guanylic acid greatly reduced tumour frequency in females but not males. Thymidine and thymidylic acid had the reverse effect. Several time-dependent features of tumorigenesis were observed: tumour formation was confined to about a 24 hr period commencing 2·5 days after emergence. All animals developing tumours died; thereafter survivors were highly resistant to tumorigenesis. Systematically ageing houseflies on a sucrose diet before feeding the TuI diet resulted in a dramatic decline in tumour frequency in females. Similarly aged males showed a high mortality rate of uncertain cause. It is suggested that tumorigenesis in adult houseflies may serve as a useful model system for the study of several aspects of insect physiology, notably nutritional balance and imbalance, mechanisms controlling the tyrosine/tyrosinase system in the blood and tissues of insects, factors influencing the darkening of the adult cuticle, ageing, pathology, and population control of insects by nutritional methods.

The oxidation of l -glycerol-1-phosphate by flight-muscle mitochondria isolated from a variety of insects has been studied. Low levels of calcium ions, 10 −7 M, and strontium ions, 10 −5 M, act as heterotropic allosteric effectors of the oxidation by both mitochondria and sub-mitochondrial particles. l -Glycerol-1-phosphate also acts as a homotropic effector in the absence of calcium and strontium ions. The preparation is described of submitochondrial particles, EGTA particles, which do not show allosteric behaviour. The site of action of calcium and strontium ions is discussed.

The dipeptide gamma- l -glutamyl- l -phenylalanine (γ-glu-phe) has been identified as a major biosynthetic product of the larva of the housefly Musca domestica L. The concentration of γ-glu-phe increases continually during larval growth, reaching a maximum of 13·9 μM/g wet wt. in the fully grown larva. More than 85 per cent of the dipeptide is found in the blood at this time. During the first few hours after the onset of hardening and darkening of the puparium, the level of γ-glu-phe falls markedly. After 8 hr of tanning virtually no trace of dipeptide can be found. The disappearance of γ-glu-phe is under the control of the moulting hormone ecdysone as shown by ligature experiments. Gamma-glu-phe may play a key role in puparium formation in the housefly by providing a large phenylalanine reservoir which can be metabolized to melanins and/or quinones for tanning cuticle proteins during sclerotization. Occurrence of γ-glu-phe appears to be restricted to the larva of flies of the genus Musca . No good a priori reason can be given for its biosynthesis by members of this genus.

Using H 3 - β -sitosterol, gas-liquid chromatographic analysis, and a semi-defined diet with aseptic rearing techniques, it was demonstrated that larvae of the houselfy, Musca domestica , do not dealkylate β -sitosterol to form cholesterol. Furthermore, the major sterol (>99 per cent) isolated from insects reared in the above manner on a diet containing pure unlabelled β -sitosterol (100 per cent by GLC) was identified as unchanged β -sitosterol. β -Sitosterol, campesterol, and cholesterol were also compared in nutritional tests in which each of these three sterols served as a sole source of sterol in both the adult and larval diets. β -Sitosterol was only approximately one-fourth as effective as either cholesterol or campesterol in supporting larval growth and development. In addition, only 1·4 per cent of the organisms from the diet containing β -sitosterol emerged as adults and these failed to produce viable eggs. These results demonstrate conclusively that β -sitosterol is not converted to cholesterol in the housefly and that this sterol will not fulfil in entirety the sterol requirement of this insect.

The addition of sterilizing doses of various chemosterilants to the food of male houseflies for 3 days following adult emergence led to a progressive decrease in content or synthesis of various constituents of the testis. As early as the fourth day of adult life, the chemosterilant damage to the testes was revealed microscopically as a shortage of spermatogonia. By the eighth to eleventh day the treatment caused deficiencies also of spermatocytes and spermatids, but not of mature sperm. These treatments reduced the uptake of radiocarbon by the testes in flies injected into the thorax 20 hr previously with a 14C-labelled protein or nucleic acid precursor. The reductions ranged up to 28 per cent when 4-day-old flies were injected and were 30–56 per cent with 11-day-old flies. Of the injected compounds, arginine-U-14C gave the highest labelling.

Thiocarbohydrazide (TCH) was the most toxic of numerous convulsants tested in houseflies and was as toxic to a DDT-resistant strain as to a susceptible strain. The LD-50 values ranged from 0·3 μ g/fly for injected aqueous TCH to 1 μ g/fly for TCH dissolved in dimethylsulphoxide and benzene and applied topically. The in vivo tracer studies with formate- 14 C revealed that both DDT and TCH increased the labelling of purines while reducing the content of radioproline. Both toxicants stimulated the production of 14 CO 2 from 14 C-labelled proline and sugars, the increase from glucose-1- 14 C being almost double that from glucose-6- 14 C. Other similar effects of the toxicants involve the metabolism of 1-carbon compounds in the presence of azaserine and urea production from arginine. Inverse metabolic effects exerted by DDT in the suceptible and resistant strains are discussed.

An oostatic hormone is produced by houseflies with eggs in stages 4 to 10. This hormone suppresses the development rate of stages 2 to 4 eggs. Complete inhibition of the second gonotropic cycle at stage 4 occurs when mature eggs from the first cycle are retained. Extracts from mature female flies inhibited ovarian development when injected into 24-hr-old flies with eggs in stage 2 of development. The oostatic hormone is soluble in 95% ethanol, chloroform-methanol (2 : 1), 20% glycerol, and water. The oostatic hormone maintains the cycle of egg production in the housefly.

Larvae of the housefly, Musca domestica, bred on chemically defined diets under aseptic conditions incorporate β-methylcholine into the phospholipids in place of choline when the dietary source of choline is replaced by either acetyl-β-methylcholine or carnitine. A low level of phosphatidylcholine is found in such larvae. Larvae fed on diets containing a β-methylcholine source pupate normally and the adults obtained retain the abnormal phospholipid when fed on glucose and water. Adult houseflies incorporate only small amounts of β-methylcholine into their phospholipids when fed on acetyl-β-methylcholine solutions. No phosphatidyl-β-methylcholine was found in adult flies fed on carnitine solutions. It appears, therefore, that the adults unlike the larvae are unable to decarboxylate carnitine. Adult houseflies bred from larvae fed on diets containing carnitine in place of choline convert most of their phosphatidyl-β-methylcholine to phosphatidylcholine when choline is available. Acetylcholine levels in the heads of adult flies bred from larvae fed on diets containing acetyl-β-methylcholine or carnitine were reduced to approximately 30 per cent of the level found in adults bred from larvae fed on diets containing choline. This compares with a reduction of the phosphatidylcholine level in the whole insect to about 6 per cent of that found in normal flies. Larvae bred on a medium containing no added choline had an increased amount of phosphatidylethanolamine and a low level of phosphatidylcholine. Larvae bred on a medium containing added monomethylaminoethanol or dimethylaminoethanol incorporate these bases into their phospholipids. Both these bases replace choline and partially replace ethanolamine in the phospholipids.

As determined by immunodiffusion and immunoelectrophoresis, the origin of the principal blood protein (Fraction 3) of newly emerged female houseflies is the larval fat body. This protein is carried over into the adult from immature stages of development. The sex-specific accumulated blood protein (Fraction 4) of milk-fed females originates in the adult fat body and is unique to the adult. The near-reciprocal relationship between the depletion of Fraction 3 and the accumulation of Fraction 4 in the blood of milk-fed females reflects the disappearance of larval fat body and formation of adult fat body, the respective sources of these blood proteins. Fraction 4 appears to arise de novo to meet requirements of the adult female, rather than being a conversion product of Fraction 3.

Methods are described for the determination of [ 14 C] acetylcholine formed in the head tissues of the adult housefly in vivo following [ 14 C] acetate injection. Acetylcholine estimated radiometrically and that assayed pharmacologically were in good agreement. This confirms that this ester largely, if not entirely, accounts for the pharmacological activity of head extracts as assayed with the frog rectus abdominis muscle preparation. The data indicated that the acetylcholine content of the brain is due to a steady state of synthesis and hydrolysis with a minimum turnover rate of 6·1 × 10 −5 μmoles acetylcholine/min/head. Turnover was apparently reduced in insects poisoned with acetylcholinesterase inhibitors. These and other observations are interpreted as showing that the ACh system of the brain is highly compartmentalized, synthesis from choline and intracellular acetate being controlled by the availability of choline arising from acetylcholinesterase action. No correlation was found between acetylcholine turnover in vivo and the incidence of light on the compound eye. Turnover was apparently unaffected by surgical elimination of possible nerve activity originating in the thoracic ganglion. It was, apparently, reduced during cyclopropane anaesthesia. Current problems of insect neurophysiology are critically discussed.

The accumulation into the nervous system of a disproportionate amount of the phosphatidylcholine present in housefly larvae when reared on diets containing very low concentrations of choline is found to occur even when two analogues of choline, N-dimethylethylcholine (DMECh) or β-methylcholine (βMCh), are included in the diet at more than one hundred times the choline concentration. Both analogues are incorporated into phospholipids of the whole larvae and nervous system. The ratio of choline to analogue found in the lipids of the whole insect is 1 : 48 (DMECh) and 1 : 24 (βMCh) and in the nervous system 1 : 22 (DMECh) and 1 : 4 (βMCh). This preferential accumulation into the lipids also occurs during the development of the adult nervous system. The total phospholipid content of the head and thoracic ganglia of the adult is two to three times that of the fused larval ganglion. This results in 40 per cent of the phosphatidylcholine present in the adult fly, obtained from larvae fed on diets containing βMCh, being concentrated in the nervous system.

The stage of ovarian development was compared with changes in the volume of abdominal fat body in female houseflies. Pupal fat body volume decreased during each stage of oogenesis and had its maximum rate of volume decreases during the vitellogenic phase of oogenesis (stages 4 to 9). No pupal fat body remained in flies with mature (stage 10) eggs. Adult fat body showed a volume increase during egg stages 2 to 7 and 9 to 10 but decreased during egg stages 7 to 9. Both ovariectomy and allatectomy resulted in a depressed rate of pupal fat body disappearance. This was attributed to the absence of functional ovaries in the allatectomized and ovariectomized females. It was hypothesized that the developing ovaries may act as a nutritional sump for vitellogenic precursors present in the fat body and/or an ovarian hormone may stimulate cytolysis of the pupal fat body. By so doing the pupal fat body volume would decrease more rapidly when developing ovaries were present. The adult fat body volume was not affected in allatectomized or in allatectomized plus ovariectomized flies, but increased in volume in ovariectomized flies. Topical applications of synthetic juvenile hormone on mature female flies resulted in an increased adult fat body volume. From this it was concluded that ovariectomy removed the inhibition on the corpus allatum and resulted in an increase in juvenile hormone titre which caused an increase in adult fat body volume.

The fine structure of flight muscle of adult houseflies (Musca domestica L.) has been investigated in order to study the changes in mitochondria and muscle cell structure with ageing. In all the specimens examined, two kinds of mitochondria are recognized: type A with simple folded cristae and a light matrix, and type B with complex multicristae surrounded by a dense matrix. However, the relative proportions of A and B, the extent of complexity of the cristae, the arrangement within the muscle fibres, and the degree to which they are surrounded with glycogen varies with age. In newly emerged flies A and B types are equally represented and are loosely arranged in between the muscle fibres and surrounded with large quantities of glycogen. About 12 days after emergence, the population of type B mitochondria increases; in some individuals the multicristae develop fenestrations and in others they become arranged as whorls resembling myelin figures. The amount of glycogen is reduced and the mitochondria are more closely packed between the muscle fibres. After 20 days the structural complexity of the mitochondria begins to decrease and degeneration starts. Type A degenerate by swelling due to non-uniform increase of their intracristal spaces, whereas type B degenerate by developing structureless areas in place of a dense matrix. The outer membrane in both types of mitochondria loosens up and acquires a fuzzy appearance. Along with a decline in mitochondrial number, the muscle substance also decreases in old flies. The relationship between the fine structure of mitochondria and biochemical changes recorded during the senescence of houseflies is discussed.

Corpus allatum area in the housefly, Musca domestica L., decreased before vitellogenesis occurred in the ovaries and then increased in area by stage 5 or 6 of ovarian development. Both ovariectomy and oviposition resulted in a drop in corpus allatum area. It was concluded that a small corpus allatum released juvenile hormone, whereas a large gland stored hormone. Injection of extract containing oostatic hormone into ovariectomized flies increased the area of the corpus allatum; 26 μg of extract maintained a large corpus allatum for 32 hr. A humorally mediated inhibitory feedback mechanism exists between the ovaries and the corpus allatum.

αMDH, a potent decarboxylase inhibitor, prevents hardening and darkening of the cuticle of adult flies when injected soon after eclosion. This effect is accompanied by an accumulation of DOPA. If the release of bursicon from the CNS is blocked no DOPA accumulates. It would appear, therefore, that one of the actions of bursicon in the regulation of adult tanning is involved with the control of the activation of tyrosine hydroxylation.

The male accessory material of houseflies is produced by what appears to be one type of cell in the anterior part of the ejaculatory duct. The cells of this part of the ejaculatory duct have an extensive rough endoplasmic reticulum, numerous free ribosomes, and many Golgi complexes. Secretion of the accessory material seems to occur as a mixture of release via individual Golgi vesicles, the splitting off of portions of cytoplasm, and the lysis of entire cells. No evidence was found of replacement of lysed cells. Synthesis of the secretory material is most active soon after emergence and after mating. The material accumulates around the duct lumen between the secretory cells and the intima and passes directly through the intima in being discharged from its storage area.

Results on the mode of recovery of inhibited cholinesterase both in American cockroaches and house flies were found indicative of a predominant recovery process that was not influenced by inhibitors which resulted in dimethyl, di-n-propyl, diisopropyl, and di-n-butyl phosphorylated cholinesterase. A study was made of both in vivo and in vitro ageing of dimethyl phosphorylated cholinesterase. The slow in vivo ageing rate found suggests that a considerable portion of non-aged dimethyl phosphorylated cholinesterase was present even during the prolonged slow recovery of cholinesterase leves. The in vivo ageing experiments combined with the slow in vivo recovery observed in other experiments can best be explained by concluding that the slow recovery is a result of cholinesterase synthesis and not a reversal of inhibited cholinesterase.

Larvae of the housefly, Musca domestica L., reared aseptically on a semi-defined diet failed to develop when either cholestenone, cholestanone, or cholestanol was used as the sole source of dietary steroid. However, when these three steroids were tested for ‘sparing’ activity (in combination with a subminimal quantity of cholesterol) cholestanone and cholestanol were 66 and 79 per cent, respectively, as effective as an optimum concentration of cholesterol. Under the same conditions cholestenone showed only low biological activity (11 per cent). Biochemical studies with 4-14C-cholestanone indicate that the utilization of this steroid in the housefly larvae proceeds through its partial conversion to cholestanol. The identity of cholestanol as a metabolite of cholestanone was established by radiotracer techniques, gas-liquid chromatographic analysis, and by isolation of crystalline cholestanol from housefly pupae reared aseptically on a semi-defined diet containing cholestanone plus a subminimal amount of cholesterol. Using reverse isotope dilution techniques, no significant conversion of 14C-cholestanone to cholesterol was detected. In studies with adult insects, both males and females converted injected 14C-cholestanone to cholestanol.

The ultrastructure of abdomen, gut, and fat body microsomal preparations from adult Musca domestica and larval Prodenia eridania was examined by electron microscopy. The components of these enzyme preparations are similar to preparations from mammalian liver and contain membranous vesicles, broken membranes, and RNP particles. Clusters of glycogen granules are a prominent feature of the Prodenia gut preparations. Microbial contaminants are retained in pellets from gut and fat body homogenates. A preliminary centrifugation at 10,000 g for 15 min which removed all mitochondria from M. domestica preparations was inadequate for this purpose when Prodenia gut was being used. The fine structure was intact in Musca and Prodenia gut microsomal pellets. The structural integrity of fat body microsomes was impaired compared to those from the gut when both preparations were made in Millonig's sodium phosphate-sucrose buffer. This difference between Prodenia gut and fat body tissues was increased further when potassium phosphate buffer was the homogenizing medium. It is apparent that buffering systems and centrifugation speeds must be varied from one insect to another and from one tissue to another of the same insect.

Centrifugation of housefly head homogenates at 105,000 g for 1 hr sediments 73 per cent of the cholinesterase (ChE). This soluble-particulate distribution was not altered by dialysis or the addition of salts and it remained the same at pH values of 7, 8, and 9. At pH 5 all of the enzyme precipitated. All of the particulate enzyme was reversibly solubilized by sodium taurocholate, and 66 per cent of it was solubilized by autolysis. Based upon gel filtration in Sephadex G-200 the autolytically solubilized enzyme has the same molecular weight as the major fraction of natively soluble ChE. The sodium taurocholate solubilized enzyme, which is presumably a taurocholate-ChE complex, is excluded from Sephadex G-200.

RNA synthesis in the developing ovary of the housefly was studied by determining nucleic acid concentration and isotopic incorporation into the purines of RNA. A method is described for the isolation and purification of cytoplasmic RNA. The base composition of the RNA was DNA-like, with a low (G+C) (A+U) ratio. Administration of 14C-labelled formate to female houseflies leads to extensive incorporation of 14C into adenine and guanine moieties of RNA in the early stages of ovarian development. Incorporation of the isotope into ovarian RNA reached a maximum at 12 hr after its administration.

The only soluble metabolite of 14 C-tyrosine injected into flies as they emerge from their puparia is tyrosine-O-phosphate. A kinetic study of its turnover in the presence and absence of the adult tanning hormone, bursicon, indicates that it is probably not an intermediate in the tanning process. Its role may be that of a store of metabolically accessible tyrosine. However, since the specific activity of tyrosine falls during the first 5 hr of adult life after emergence and there is insufficient tyrosine phosphate to account for the observed dilution rates, it is suggested that a proteinaceous or peptide precursor is degraded at this time. The parallel dilution rate of 14 C lysine lends support to this possiblity.

Mating in the housefly, Musca domestica (L.), was correlated with the stage of ovarian development. Females with stage 3 and 4 ovaries rarely mated, but mating increased from 29·0 per cent in females with stage 5 ovaries to 97·0 per cent in females with stage 10 ovaries. When they were given a choice, males preferred females with ovaries in stages 6 to 10 for mating. Females that were allatectomized when they were 48 hr old or younger demonstrated both an inhibition of ovarian development and mating. The corpus allatum was activated in 100 per cent of the female population held at 21·7 ± 1°C after 60 hr. Ovariectomized females mated normally but tended to remate more frequently than the controls. Since topical application of 2·5 μg of the synthetic juvenile hormone stimulated mating and ovarian development, it was concluded that juvenile hormone stimulated both ovarian maturation and mating in the housefly and that the correlation between mating and ovarian development was caused by a response to the titre of this hormone.

The toxicities of diazinon and diazoxon to two strains of housefly (normal and resistant) were measured by injection and topical application. Both compounds were more toxic by injection. The difference in susceptibility by the two ways of poisoning was greater with the resistant than the susceptible flies because insecticide penetrates less readily into the resistant flies, as indicated by measurement of loss from the surface using radioactive diazinon. Resistance of both strains increased with age; C 14 diazinon penetrated the cuticle of younger flies more rapidly than of older flies. Even when injected, more diazinon or diazoxon was needed to kill resistant flies than the susceptible, indicating that penetration was not the only reason for resistance. C 14 diazinon was decomposed more efficiently by resistant than by susceptible flies.

The fatty acid composition of the phospholipids and triglycerides extracted from housefly larvae reared on diets containing no added fatty acids but containing differing concentrations of choline has been determined. Reducing the choline content of the diet resulted in a graded reduction of the percentage of phosphatidylcholine present in the phospholipids of the larvae. This was accompanied by changes in the fatty acid composition, choline deficiency causing an increased utilization of 16-C rather than 18-C acids by the phospholipids. Changes in the fatty acid composition of the triglyceride fraction were also observed but these were associated with insects containing very low levels of phosphatidylcholine. Examination of the fatty acids in the different classes of phospholipids showed that the major change resulting from choline deficiency was in the fatty acids of the phosphatidylethanolamine fraction—the phospholipid which increased as the phosphatidylcholine decreased. Although the fatty acid composition of the different classes of phospholipids was not completely fixed, some preferential utilization of certain fatty acids by certain classes was observed, in both larval and adult insects. The fatty acid composition of the phospholipids extracted from larval gut, muscle, fat body, cuticle, trachea, nervous tissue, and haemolymph was determined. Changes resulting from choline deficiency similar to those seen in the whole larva were observed in all tissues except the nervous tissue. The effect of rearing larvae at temperatures between 24 and 35°C resulted in only minor changes in the fatty acid composition of both phospholipid and triglyceride fractions but the difference due to choline deficiency was observed at all temperatures. The possibility that the observed changes in the fatty acids of the phospholipids are compensatory to the changes in the proportion of the choline to the ethanolamine phospholipids is discussed.

Transmission electron microscopy of the first optic ganglion of the house fly Musca domestica reveals that retinular (R) axons branch and interdigitate throughout the external plexiform layer. Axonal membranes of neighboring cells are in close apposition to each other, usually without junctional modifications. In some cases R axonal membranes exhibit greater electron density in the areas of contiguity. Adjacent axons may show a near confluency of axoplasm at points where membrane boundaries are interrupted or obscured. Physiological implications of these ultrastructural findings are discussed.

The endocrine system involved in the control of oogenesis in the housefly, Musca domestica vicina , was investigated. Allatectomy, decapitation, and starvation of newly emerged females resulted in inhibition of oogenesis, showing a close relationship between enlargement of the corpus allatum and growth of follicles during the first oogenesis. Histological observation of sexually matured females showed active secretion of the corpus allatum and the medial neurosecretory cells of pars intercerebralis. Topical application of juvenile hormone analogues (JHA) to the allatectomized fly induced the growth of ovary, and critical doses of methoprene and methyl-7, 11-diethyl-juvenate for the maturation of the ovary were determined. JHA stimulated initiationof oogenesis in the starved or decapitated flies as well as vitellogenesis in the sugar-fed one; subsequently it was found that juvenile hormone acted not only as a gonadotropin but also as a regulator of vitellogenesis. Furthermore, JHA stimulated cell lysis in pupal fat body of female flies, indicating a possible influence of juvenile hormone upon the process of releasing vitellogenin.

The housefly larvae were grown in the aseptic diet containing 0.56 μmole cholesterol/g wet weight of diet (control) and 0.05 μmole cholesterol/g wet weight of diet (deficient). The effects of cholesterol deficiency upon the phospholipid composition and fatty acids of the total phospholipid and triglyceride fractions from the lipid extract of the various larval tissues, whole larva, and in both sexes of adults 4 days after eclosion were examined. The total sterol and phospholipid contents (expressed relative to the wet weight of the insect) of the control and deficient insects at the larval and adult stages were analysed and molar ratios compared. The results suggest that cholesterol deficiency reduced the free sterol content of the larvae and adult insects to approximately 25% of the content of the control insects. However, cholesterol deficiency did not effect the phospholipid content during larval and adult stages when compared to that of control insects. Though the larvae reared on the cholesterol deficient diets did not show a profound alteration in the phospholipid composition, a marked increase in the ratio of phosphatidylcholine to phosphatidylethanolamine of the larval fat body and composite gut fraction were noticed. The cholesterol deficiency induced significant changes in the fatty acid composition of the phospholipid fraction of the insect. The ratio of unsaturated fatty acids to saturated fatty acids of the phospholipid fractions decreased significantly due to cholesterol deficiency in the whole larvae and in both sexes of adult flies. The data indicates that cholesterol deficient insects compensated for the lack of cholesterol by increasing saturated fatty acids preferentially in the phospholipid fraction of the lipids for the maintenance of proper membrane fluidity.

Thirty days after primary cultures were made of dispersed embryonic cells of the housefly, Musca domestica, large numbers of dividing undifferentiated cells were present. The cells were subcultured. After three passages, the doubling time had stabilized so that subcultures could be made at 7 to 10 day intervals. When subcultured, the cells attached and grew on the surface of the flasks and formed dense cell sheets and clumps. The derived cell line has been maintained for over 2 years, and the nutritional requirements have been studied. Cytogenic studies of the line at passages 14, 27, and 48 showed that the line has a chromosome count of 11 (2n = 12) and the missing chromosome is probably the Y chromosome. Thus we believe that the line is fundamentally diploid.

The effect of various concentrations of Altosid and actinomycin D under defined conditions on housefly metamorphosis was investigated with three strains of houseflies. The morphogenetic response varied with the strains and the length of time which the larvae were exposed to the juvenile hormone analogue. De novo RNA synthesis was studied with (2-14C)-glycine. Methods were developed for the isolation of nuclear, soluble, and ribosomal RNA. The procedure presented provides a DEAE-cellulose chromatographic method for the removal of high molecular weight RNA from DEAE at a neutral pH. Labelling of the RNAs was increased in the presence of the juvenile hormone analogue indicating an increase in the rate of RNA synthesis. The higher incorporation of the labelled precursor into nuclear RNA demonstrates that cytoplasmic RNA is derived from the nuclei.

Housefly larvae and adults of the Isolan-B strain, insecticide resistant because of high microsomal oxidase activity, and the susceptible WHO Standard Reference (SR) strain, were reared on diets containing phenobarbital or piperonyl butoxide. Periodically throughout the treatments groups of insects were assayed for microsomal aldrin epoxidase activity. This activity was compared with such growth and development parameters as pupation, adult emergence, and reproduction in similar groups of insects. At diet levels of 0·01 to 0·50 per cent both chemicals caused large increases, as much as elevenfold in microsomal oxidase activity in third instar larvae and 15 to 100 per cent inhibition of pupation and emergence. In the adult diet at 1 per cent, both compounds caused at least 50 per cent decrease in egg production. Phenobarbital enhanced the enzyme system in both larval and adult stages of both strains but piperonyl butoxide, while enhancing enzyme activity in larvae of the Isolan-B strain, was an inhibitor in the adult stage. The results are interpreted as an indication of a direct connexion between microsomal oxidase activity and the action of hormones in the housefly, probably through regulation of hormone titre by these enzymes.

Abdomens of housefly larvae which are isolated before pupariation and injected with labelled or unlabelled cholesterol are shown by bioassay, thin-layer and gas-liquid chromatography, and tracer experiments to synthesize α- and β-ecdysone. Autoradiography of cross-sections of abdomens injected with labelled cholesterol show that oenocytes are intensively labelled. The results are discussed with respect to the validity of the Calliphora or Musca assay for ecdysones.

When the female housefly retains eggs, vitellogenesis in the penultimate oocytes is suppressed during continued protein feeding. Allatectomy of gravid females or of females with developing oocytes did not prevent maturation of a second batch of eggs. This result does not support the claim of Adams (1970) that an oostatic hormone, produced by ovaries with retained eggs, inactivates the corpus allatum (CA) and thereby prevents development of the next batch of eggs. The report of Adams and Hintz (1969) that the CA regulates egg maturation depends on their removal of ring gland, which they incorrectly refer to as ‘allatectomy’. In the present report, removal of the ring gland from 1 day old females suppressed egg development, whereas removal of the CA alone did not. Therefore, sufficient CA hormone for reproduction was secreted within 24 hr of emergence, and it was the removal of the corpus cardiacum, and not the CA, that had prevented egg development in the experiments of Adams and Hintz.

Quantitative analyses have been made of the dietary cholesterol requirement for the growth of the larvae of Musca domestica . The larvae will not grow on diets to which no cholesterol is added, a few pupae and adults are obtained when the concentration of cholesterol is 0·05 μmol/g of diet, but the concentration has to be raised to 0·36 μmol/g of diet before the maximum numbers of pupae and adults are obtained. Further addition of cholesterol above 0·36 μmol/g diet did not have any significant effect on the weight and growth of the larvae. However, the ratios of the cholesterol to phospholipid fractions recovered from the larvae increased rapidly when the concentration of cholesterol in the diet was raised from 0·05 to 0·56 μmol/g of diet. Above this concentration only a slight increase in the ratios was observed. Larvae reared on diets containing 0·05 μmol cholesterol/g of diet contain only 25 per cent of the cholesterol content of the larvae reared on the diets containing more than 0·28 μmol of cholesterol/g of diet, the cholesterol content being expressed relative to the weight of the larvae, The absence of cholesterol synthesis has been demonstrated in the larvae by feeding [4- 14 C] cholesterol. The specific activity of the cholesterol recovered from the larvae is the same as that of cholesterol added to the diet. Irrespective of the cholesterol concentration of the larval diet, approximately 97 per cent of the radioactivity recovered from the larvae behaved as free cholesterol, less than 1 per cent as cholesterol esters and the rest as unidentified ‘polar sterols’. The results are compared with those from similar studies on other insects.

β-Alanine synthesis has been studied in the housefly, Musca domestica. The larvae were reared aseptically on a β-alanine free diet and upon pupation were injected with uracil-6-3H, aspartate-U-14C, pantothenate-1-14C, propionate-2-14C, or malonate-2-14C. It was found by radioassay that relative to the other compounds tested uracil contributed 56·2 per cent and aspartate 24·2 per cent, while pantothenate, propionate, and malonate contributed 9·4, 7·1, and 3·1 per cent, respectively, to β-alanine synthesis.

Adults of the housefly, Musca domestica , and the cockroach, Periplaneta americana , were acclimated at high and low temperatures. Apyrase activity determinations were then made from housefly thoracic and cockroach coxal muscles. At given temperatures the apyrase activity rates were higher and temperature coefficients were lower in cold- than in warm-acclimated insects. The number of mitochondria was greater in muscle from cold-acclimated individuals. The results are discussed in terms of their significance in thermal compensation in insects.

Oogenesis in the housefly, Musca domestica , was divided into a series of 10 stages where stage 1 was the germarium, stage 4 was the beginning of yolk deposition, stage 7 was characterized by maximal nurse cell development, stage 9 by the degeneration of the nurse cells and chorion formation, and stage 10 was the mature egg. It required 69 hr from eclosion at 27°C to develop mature eggs. This represented an oocyte volume increase of 3700-fold, a seventeenfold increase in follicle length, and a sevenfold increase in weight. The application of 2 μg of isopropyl (E,E)-11-methoxy-3,7,11-trimethyldodeca-2,4-dienoate (ZR-515) to allatectomized (-CA) flies stimulated egg development, which progressed at the same rate as the controls. The -CA flies did not develop eggs past stage 4, which represented a cessation of development at a volume of 1·4 per cent that of a mature egg and an ovarian dry weight of 11 per cent that of a mature ovary. The follicle cells from -CA flies did not differentiate into the squamous condition over the nurse chamber, did not become columnar over the oocyte, did not produce the chorion or vitelline membrane, and did not decrease in number as they did on the stage 10 follicles. Endomitosis in the nurse cell nuclei of -CA flies stopped development at 290 c, but maximum development of 2400 c occurred in stage 7 follicles from controls, and then the nurse cells began to disintegrate.

Qualitative and quantitative analyses have been made of the phospholipid composition of haemolymph from larvae of Musca domestica and of the two lipoprotein fractions separated from it by agarose-gel electrophoresis. The effect of rearing the larvae on defined diets containing adequate choline, no added choline, and choline plus 2-aminobutan-1-ol on this composition has been studied. The haemolymph lipoproteins have a phospholipid pattern similar to that of the unfractionated haemolymph. The chief component is phosphatidylethanolamine. Diglycerides and free sterols are the major neutral lipids present in the haemolymph and the separated lipoproteins. The different diets cause changes in the various phospholipids present in the two lipoproteins which are similar to those that occur in other tissues of the larvae. Choline deficiency increased the proportion of the haemolymph phospholipid that is associated with the lipoprotein having the slower electrophoretic mobility. The results are compared with those obtained from other insects and vertebrates and the role of the lipoprotein in choline deficiency in the housefly is discussed.

The amount of in vivo thoracic cholinesterase inhibited in houseflies at the time of knock-down was determined after the application of six different organophosphate anticholinesterases. For each compound, a specific level of inhibition was required for knock-down. The data clearly show that, when comparing the effects of different inhibitors, there was no one level of total cholinesterase inhibition which resulted in poisoning symptoms. The inhibition level at knock-down appeared to be inversely related to the reactivity of the organophosphate.