Rapid in vitro activation of corpora allata by extracted locust brain allatotropic factor

Brains of young (newly emerged) adult female locusts (Locusta migratoria migratorioides) and of mature (> 9 days old) locusts contain an extractable allatotropic factor, soluble in 100% methanol and in distilled water. This factor stimulates juvenile hormone III (JH III) synthesis and release from corpora allata (CA) that have been excised from donor locusts and then incubated with (radiolabeled methyl)-methionine in vitro in its presence. In addition to JH III, which is the major product synthesized by the CA, other hexanesoluble, radiolabeled compounds–-more polar than JH III–-are also released when CA are incubated in vitro. The activation of CA by the allatotropic factor is rapid and quickly declines when the factor is removed from the medium. Corpora allata excised from young females are marginally active and can be activated by brain allatotropic factor to less of an extent than CA of mature locusts. The content of allatotropic factor in brains of mature locusts is higher than that ascertained in brains of young females. Allatotropic factor is also present in the corpora cardiaca.     

Étude du contrôle neuro-endocrine du fonctionnement du corpus allatum chez les larves du quatrième stade de Rhodnius prolixus

This paper concerns the effects of electrocauterization of the pars intercerebralis (P.I.) in the penultimate instar (fourth) of Rhodnius prolixus performed at various intervals following a blood meal. The following ecdysone-induced apolysis is strongly adultoid in the larvae whose P.I. was destroyed within 20 hr after a meal. The same operation at 24 hr, 48 hr, and 7 day intervals accompanied with ecdysone injections does not produce such effects since the resulting cuticle synthesis is normal (larval). These experiments demonstrate that the protocerebrum has an allatotropic action during the 20 hr following the blood meal. The stimulation of the corpus allatum is effected by a humoral pathway as shown by experiments of disconnecting C.A., cauterization of the P.I., and the application of juvenile hormone. Allatotropic and prothoractotropic effects of the brain cannot be induced by implanting entire brains or only P.I. The results of parabiosis between a fed allatectomized insect and a parsectomized one just after a meal do not allow us to evaluate the allatotropic effects as the apolysis does not occur in such pairs.These results have permitted us to discuss the secretory activity of the brain and its interactions with other endocrine glands.     

Hormonal regulation of trehalose metabolism in the blowfly, Phormia regina: interaction between hypertrehalosemic and hypotrehalosemic hormones

Hypertrehalosemia occurs two days after cardiacectomy of adult male Phormia regina with no attendant change in fat body glycogen. In spite of this, cardiacectomized flies caused to fly for 10 min show a lower rate of haemolymph trehalose turnover, and seem to have a decreased capability for synthesizing trehalose from haemolymph glucose. Phormia brain is shown to contain a hypotrehalosemic hormone whose release depends on the integrity of the stomatogastric nervous system. It is possible that the hypertrehalosemic condition in cardiacectomized flies is a result of the absence of this hormone from the blood.     

The role of allatostatic and allatotropic neuropeptides in the regulation of juvenile hormone biosynthesis in Lacanobia oleracea (Lepidoptera: Noctuidae)

In the sphinghid moth Manduca sexta, two allatoactive neuropeptides appear to be responsible for regulating juvenile hormone (JH) production by the corpora allata (CA). These peptides (M. sexta allatostatin, Mas-AS, and M. sexta allatotropin, Mas-AT) respectively inhibit and stimulate in vitro JH biosynthesis by CA in this insect. However, although Mas-AS inhibits CA in both larval and adult insects, Mas-AT is active only in adult M. sexta. The situation in other lepidopteran species is less clear-cut and, although both peptides have been detected (usually by immunologic and/or molecular techniques) in several other moths (including noctuids), their function as regulators of JH production remains uncertain. In the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae), we have previously demonstrated the occurrence of Mas-AS and/or Mas-AT in extracts of CA, brain and other organs, and have shown that both peptides are present in larval and adult forms. However, in L. oleracea, although Mas-AS inhibits larval and adult CA in vitro, it does so only at relatively high concentrations, and to a maximum of only approximately 70%. By contrast, Mas-AT (which is also present in larval and adult L. oleracea) stimulates larval and adult CA, but is substantially more potent ( approximately 100 fold) than the allatostatin. In this paper we present the results of paired, concurrent measurements (using ELISA) of levels of Mas-AS and Mas-AT in brains, CA and hemolymph (plasma and hemocytes) of L. oleracea at times when there are marked changes in JH titers. We also present data on the in vitro rates of JH biosynthesis by isolated CA, and on hemolymph JH esterase activity measured at the same critical developmental times, and discuss all of these data in relation to the putative allatoregulatory roles of the M. sexta allatotropic and allatostatic neuropeptides in L. oleracea.     

Effect of lighting conditions on endocrine events in Galleria mellonella

Galleria mellonella larvae reared in a light:dark (LD) 12:12 regime terminate feeding and pupate 1 day earlier than insects kept in constant darkness. Rearing conditions have no influence on the body weight attained at pupation. In both rearing conditions body weights attained by females were greater on the average by 50% than the body weights of males.In LD 12:12 all distinctive changes in the juvenile hormone titre and juvenile hormone esterase activity occurred 1 day earlier than in constant darkness. The peak value of juvenile hormone esterase activity was by 22% higher in insects reared under light conditions as compared with animals kept in constant darkness.Last-instar G. mellonella larvae reared in constant darkness were more sensitive to cooling and juvenile hormone analogue application. The chilling-induced elevation of juvenile hormone titre was also higher in constant darkness. The chilling-induced elevation of the brain allatotropic activity was not influenced by rearing conditions (brain activity measured as the number of extra-larval moults produced by hosts implanted with chilled brains). However, rearing in an LD 12:12 regime decreases the host sensitivity to implanted brains.     

Age of three dipteran hosts as a factor governing the pathogenicity of Beauveria bassiana and Metarrhizium anisopliae

The age of three adult dipteran hosts was tested as a factor governing the virulence of two fungal pathogens. The subjects tested were 1-, 5-, 10-, 15-, 20-, 25-, and 30-day-old animals. The age of the house fly, Musca domestica, the age of the black blowfly, Phormia regina, and the age of the onion fly, Hylemya antiqua, do not significantly affect the virulence of either Beauveria bassiana or Metarrhizium anisopliae. B. bassiana kills M. domestica and P. regina more quickly than does M. anisopliae, but B. bassiana and M. anisopliae kill H. antiqua at the same rate. The ages of all three flies had no significant effect on average death time after inoculation with either pathogen.     

Serotonin Inhibits Protein Feeding in the Blow Fly, Phormia regina (Meigen)

Serotonin is an important signaling molecule involved in the control of feeding in flies and other animals. In this study, a potential neurohemal release site for serotonin and the effects of exogenous serotonin on protein feeding were examined in the black blow fly, Phormia regina. A dense network of varicose neural processes exhibiting serotonin-like immunoreactivity was identified on the dorsal region of the thoracico-abdominal ganglion in P. regina. This dorsal region of the central nervous system is a likely site for the release of serotonin into the hemolymph. Circulating serotonin may have multiple systemic effects on fly physiology, including modulating or regulating feeding related processes and diuresis. Injections of exogenous serotonin reduced protein meal size in female flies at all doses and at all time points examined within a 24 h period relative to control and saline injected flies. Similar results were observed in serotonin-injected males at 35 min post injection. The injection of 50 μg serotonin resulted in the greatest amount of protein feeding inhibition. Serotonin injected flies also experienced greater weight loss than control or saline-injected flies during the 24 h post-injection period, possibly due to increased diuresis.     

Allatotropic and allatostatic activities in extracts of brain and the effects of Manduca sexta allatotropin and Manduca sexta allatostatin on juvenile hormone synthesis in vitro by corpora allata from the Eri silkworm, Samia cynthia ricini

Both allatotropic and allatostatic activities were found in crude extracts of brain from adult and larval Eri silkworm, Samia cynthia ricini, but it seems that allatotropic activity dominates in each stage. There was a high level of allatotropic activity in the crude extract of brain from newly emerged female adults, but allatostatic activity appeared in the bioassay when excessive amounts of crude extracts of brain were added. Crude extracts of brain from premoulting fourth‐instar larvae and from newly ecdysed fifth‐instar larvae exhibited allatotropic activities, whereas extracts of brain from the second and third day of the fifth‐instar larvae inhibited juvenile hormone (JH) release slightly. Allatotropic activity from the brains of adults and larvae stimulated both adult and larval corpora allata (CA) to synthesize JH. Manduca sexta allatotropin (AT) (Mas‐AT) and M. sexta allatostatin (AST) (Mas‐AST) also stimulated and inhibited both adult and larval S. cynthia ricini CA to synthesize JH, respectively. Higher concentrations of Mas‐AT (10^?4 or 10^?3 M) showed an inhibitory effect on adult CA. CA from newly emerged female adults were the most sensitive to inhibition by Mas‐AST, whereas CA from female pharate adults at about 6 h before adult emergence were the most sensitive to stimulation by Mas‐AT and S. cynthia ricini brain allatotropic activity. An extract of brain and Mas‐AT induced some of the non‐active female pharate adult CA at 12 h before emergence to synthesize a small amount of JH.     

In vitro bioassay for the brain allatotropic hormone of Galleria mellonella (Lepidoptera).

An in vitro sensitive bioassay for the Galleria mellonella brain allatotropic hormone (ATTH) was developed. This assay measures the rate of juvenile hormone (JH) synthesis in corpora cardiacacorpora allata complex (CC-CA) stimulated in vitro by ATTH released from the brain during short-term in vitro incubation, or by ATTH extracted from the tissue with methanol. CC-CA of the late VIth instar (VI3) larvae were used for assessment of ATTH. The maximum activation of test CC-CA by ATTH occurred at a concentration of 2 brain equivalents (per 100 ul medium). The highest ATTH activity was exhibited by the brains of chilled VII1 larvae: ATTH extracted from freshly dissected brains, or ATTH released from these brains during 6 h in vitro incubation, activated JH synthesis in the CC-CA nearly five or four times, respectively. The brain of VII1 hydroprenetreated larvae were ATTH inactive.     

DNA barcoding and the differentiation between North American and West European Phormia regina (Diptera,?Calliphoridae,Chrysomyinae)

Phormia regina (the black fly) is a common Holarctic blow fly species which serves as a primary indicator taxon to estimate minimal post mortem intervals. It is also a major research model in physiological and neurological studies on insect feeding. Previous studies have shown a sequence divergence of up to 4.3% in the mitochondrial COI gene between W European and N American P. regina populations. Here, we DNA barcoded P. regina specimens from six N American and 17 W European populations and confirmed a mean sequence divergence of ca. 4% between the populations of the two continents, while sequence divergence within each continent was a ten-fold lower. Comparable mean mtDNA sequence divergences were observed for COII (3.7%) and cyt b (5.3%), but mean divergence was lower for 16S (0.4–0.6%). Intercontinental divergence at nuclear DNA was very low (≤ 0.1% for both 28S and ITS2), and we did not detect any morphological differentiation between N American and W European specimens. Therefore, we consider the strong differentiation at COI, COII and cyt b as intraspecific mtDNA sequence divergence that should be taken into account when using P. regina in forensic casework or experimental research.     

Do the brains of wax moth larvae secrete an allatotropic hormone?

The hypothesis that the brains of young, last instar larvae of Galleria mellonella (L.) initiate supernumerary larval apolyses by secreting an ‘allatotropic factor’ was reexamined. It was confirmed that following bilateral allatectomy the larvae lose their ability to produce supernumerary instars (superlarvae) in response to implanted brains. The JH analog Altosid caused the allatectomized larvae to undergo extra apolyses irrespective of whether or not brains had been implanted. Although the percentage of superlarvae obtained following Altosid treatment was not increased by the implanted brains, the onset of extra apolyses was accelerated. This suggests that the brain can promote larval-larval apolyses without acting first on the corpora allata (CA). Presumably, it does so by producing prothoracotropic hormone.The propensity to generate new larval structures was tested by injecting ecdysterone into larvae 48 and 65 hr after they had been allatectomized. Within 48 hr after both CA had been removed the precocious apolysis resulted in individuals with antennae that were partly larval and partly pupal, and by 65 hr the ability to reproduce larval parts had diminished further. Those that were hemi-allatectomized did not demonstrate this impairment. The results were consistent with the interpretation that allatectomy abolishes the capacity to produce superlarvae because the JH titer declines to a level insufficient to permit expression of the larval genetic program during the next moulting cycle. This is offered as an alternative to the hypothesis that allatectomy prevents implanted brains from producing superlarvae because the target organs of the ‘allatotropic factor’ have been removed.An attempt was made to confirm the observation that brains from young, last instar larvae are more effective initiators of supernumerary apolyses than those from donors in the process of pupating. There was no evidence for a different endocrine function by the brain during the two stages.     

Blood meal as a regulator of triacylglycerol synthesis in the haematophagous stable fly,Stomoxys calcitrans

Summary Accumulation of triacylglycerol and glycogen reserves following meals of blood and/or sugar-water was investigated in the haematophagous stable fly,Stomoxys calcitrans. In the starved fly, triacylglycerol reserves appear to be utilized predominantly for energy requirements. When the starved flies were fed one meal of 1.0 M sucrose-water, they accumulated considerable amounts of glycogen but there was no increase in the triacylglycerol content. Starved flies fed one meal of blood accumulated large quantities of triacylglycerol but no glycogen; in those flies fed sugar-water and blood, glycogen and triacylglycerol accumulated. This study shows that the stable fly preferentially utilizes blood for triacylglycerol synthesis and sugar for glycogen synthesis. It is suggested that one or more factor(s) in the blood meal influences accumulation of triacylglycerol in this insect, possibly through an hormone from the corpora cardiaca-corpora allata complex.     

Culture of Entomophthora muscae in vivo and its infectivity for six species of muscoid flies

A method for the continuous culture of Entomophthora muscae (Entomophthoraceae) in adult house flies (Musca domestica) is described. Using this method we have maintained the host-pathogen system in the laboratory for more than one year. The ability of this isolate to cause fatal infections in virtually all of the house flies at risk has remained constant. The face fly (Musca autumnalis), the onion fly (Hylemya antiqua), and the seed-corn fly (H. platura) are susceptible to this isolate. The stable fly (Stomoxys calcitrans), the black blow fly (Phormia regina), and the false stable fly (Muscina stabulans) are not.     

Dosage effect study of the microsporidian Octosporea muscaedomesticae in the adult black blowfly Phormia regina,with reference to the reproductive cycle of the pathogen

The quantitative pathogenicity of the microsporidian Octosporea muscaedomesticae in adult Phormia regina was studied. Dosage levels ranging from 10² to 10⁶ spores per fly were administered to five and six groups of newly emerged, starved adult flies in two trials. Rates of mortality and infection were determined. A direct relationship between number of spores ingested and subsequent infection rate was found in a 4-day trial while no such relationship was found in an 18-day trial, using the same source of inoculum and host flies from the same colony. The lack of a direct relationship between spore dose and rate of infection in the 18-day trial is explained on the basis of the short spore-to-spore development time of the parasite. New generations of spores formed within the host tissues obscure the results in relation to the spore dose initially administered. An appreciable number of spores in the inoculum is needed to initiate frank infection. The ID₅₀ (median infective dose) was 4.4 × 10⁴ spores per fly after 4 days.     

Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease

The accumulation and aggregation of phosphorylated tau proteins in the brain are the hallmarks for the onset of Alzheimer's disease (AD). In addition, disruptions in circadian rhythms (CRs) with altered sleep-wake cycles, dysregulation of locomotion, and increased memory defects have been reported in patients with AD. Drosophila flies that have an overexpression of human tau protein in neurons exhibit most of the symptoms of human patients with AD, including locomotion defects and neurodegeneration. Using the fly model for tauopathy/AD, we investigated the effects of an exposure to dim light at night on AD symptoms. We used a light intensity of 10 lux, which is considered the lower limit of light pollution in many countries. After the tauopathy flies were exposed to the dim light at night for 3 days, the flies showed disrupted CRs, altered sleep-wake cycles due to increased pTau proteins and neurodegeneration, in the brains of the AD flies. The results indicate that the nighttime exposure of tauopathy/AD model Drosophila flies to dim light disrupted CR and sleep-wake behavior and promoted neurodegeneration.     

Allatotropic and nervous control of corpora allata in the adult male loreyi leafworm, Mythimna loreyi (Lepidoptera: Noctuidae)

Allatotropic activity is found in methanolic extracts of the brain–suboesophageal ganglion (SOG)–corpora cardiaca (CC) complex from virgin males of Mythimna loreyi Duponchel (Lepidoptera, Noctuidae). Corpora allata (CA) from 6‐day‐old virgin males exhibit low rates of release of Juvenile Hormone (JH) acid (JHA) in vitro. Release of JHA can be activated by the addition of an extract of brain–SOG–CC complex in a dose‐dependent manner, and this allatotropic activation can be sustained consistently in the continuous presence of such extracts. Based on its trypsin sensitivity and heat stability, the allatotropic factor is most likely a peptide. The allatotropic activity is dependent on the concentration of calcium ions in the medium, with the highest activation achieved beyond 2 m m . The results of nerve transection experiments suggest that both nervi corporis allati I (NCA I) and NCA II are involved in mediating the allatotropic control of CA in vitro. Isolated CA alone show significantly higher rates of release of JHA than the intact brain–SOG–CC–CA complex during the first 3 h of incubation, but the release of JHA reaches almost the same range in both groups by the end of the fourth hour of incubation.     

SIFamide and SIFamide Receptor Define a Novel Neuropeptide Signaling to Promote Sleep in Drosophila

SIFamide receptor (SIFR) is a Drosophila G protein-coupled receptor for the neuropeptide SIFamide (SIFa). Although the sequence and spatial expression of SIFa are evolutionarily conserved among insect species, the physiological function of SIFa/SIFR signaling remains elusive. Here, we provide genetic evidence that SIFa and SIFR promote sleep in Drosophila. Either genetic ablation of SIFa-expressing neurons in the pars intercerebralis (PI) or pan-neuronal depletion of SIFa expression shortened baseline sleep and reduced sleep-bout length, suggesting that it caused sleep fragmentation. Consistently, RNA interference-mediated knockdown of SIFR expression caused short sleep phenotypes as observed in SIFa-ablated or depleted flies. Using a panel of neuron-specific Gal4 drivers, we further mapped SIFR effects to subsets of PI neurons. Taken together, these results reveal a novel physiological role of the neuropeptide SIFa/SIFR pathway to regulate sleep through sleep-promoting neural circuits in the PI of adult fly brains.     

What determines the number of ovarioles in a fly ovary?

The relation between the size of a fly and the number of ovarioles in its ovary was investigated in Phormia regina and Sarcophaga bullata. Small flies of varying size were produced by taking larvae prematurely off the food. The smallest flies thus obtained were derived from larvae only $\text{1}\text{8}$ of the weight of a normal larva. The number of ovarioles in an ovary is directly proportional to the size of the fly and, in the extreme case, is about $\text{1}\text{5}$ the normal number in Sarcophaga and about $\text{1}\text{3}$ in Phormia. Larvae prematurely taken off the food, but fed again after starving for several days, grow to normal or almost normal size and develop ovaries with about the normal number of ovarioles. Small or re-fed Sarcophaga do not show any changes in the anatomy of individual ovarioles but in Phormia disorders in ovariole development and a consequent reduction of fertility are frequent. The number of ovarioles remains identical from the early pupal stage all through the development of the pharate fly and then through ovarian development in the adult fly: it is determined by the size of the larva when it was taken off the food. This shows that it is not lack of space in a small adult fly abdomen which determines the number nor the occurrence of degenerative processes during ovarian development.     

Proteus mirabilis interkingdom swarming signals attract blow flies

Flies transport specific bacteria with their larvae that provide a wider range of nutrients for those bacteria. Our hypothesis was that this symbiotic interaction may depend on interkingdom signaling. We obtained Proteus mirabilis from the salivary glands of the blow fly Lucilia sericata; this strain swarmed significantly and produced a strong odor that attracts blow flies. To identify the putative interkingdom signals for the bacterium and flies, we reasoned that as swarming is used by this bacterium to cover the food resource and requires bacterial signaling, the same bacterial signals used for swarming may be used to communicate with blow flies. Using transposon mutagenesis, we identified six novel genes for swarming (ureR, fis, hybG, zapB, fadE and PROSTU_03490), then, confirming our hypothesis, we discovered that fly attractants, lactic acid, phenol, NaOH, KOH and ammonia, restore swarming for cells with the swarming mutations. Hence, compounds produced by the bacterium that attract flies also are utilized for swarming. In addition, bacteria with the swarming mutation rfaL attracted fewer blow flies and reduced the number of eggs laid by the flies. Therefore, we have identified several interkingdom signals between P. mirabilis and blow flies.