Effect of diet on the neuroendocrine system and egg development in the sheep blowfly, Lucilia sericata

ABSTRACT. A light microscope study of the endocrine and ovarian systems of Lucilia sericata under two diets revealed that in young females fed on sugar and water, medial neurosecretory cells (MNC) synthesized and stored neurosecretory material (NSM) as the flies matured. The MNC remained filled with NSM as long as the diet was maintained. Following a small increase immediately after emergence, the size of the corpora allata (CA) showed little further change, and the nuclei of nurse cells remained small. However, rapid changes occurred in these tissues soon after a meat meal: NSM was discharged from the MNC, and the CA increased in size. These changes were at a maximum 20 h after a meat meal. 4h later, vitellogenesis was well established and the nurse cell nuclei had increased in size 20-fold. Growth of the nurse cell nuclei continued until approximately 6 h before the completion of vitellogenesis when they are resorbed. Oögenesis took about 48 h at 25°C. When 100 μg of each of three different juvenoids were applied topically to different sugar-fed flies, the nuclei of both MNC and nurse cells became enlarged, whereas the CA were somewhat reduced in size. The relationship between protein ingestion and oogenesis is discussed, and the results obtained with L. sericata are compared with those of other species, especially the blowfly Calliphora erythrocephala.     

Role of juvenile hormone and allatotropin on nutrient allocation, ovarian development and survivorship in mosquitoes

Teneral reserves are utilized to initiate previtellogenic ovarian development in mosquitoes. Females having emerged with low teneral reserves have reduced juvenile hormone (JH) synthesis and previtellogenic development. We investigated what role JH, allatotropin (AT) and other head-factors play in the regulation of previtellogenic ovarian development and adult survivorship. Factors from the head are essential for corpora allata (CA) activation and reproductive maturation. We have shown that decapitation of females within 9-12h after adult ecdysis prevented normal development of the previtellogenic follicles; however maximum previtellogenic ovarian development could be induced in decapitated females by topically applying a JH analog. When females were decapitated 12 or more hours after emergence nutritional resources had been committed to ovarian development and survivorship was significantly reduced. To study if allatotropin levels correlated with teneral reserves, we measured AT titers in the heads of two adult phenotypes (large and small females) generated by raising larvae under different nutritional diets. In large mosquitoes AT levels increased to a maximum of 45 fmol in day 4; in contrast, the levels of allatotropin in the heads of small mosquitoes remained below 9 fmol during the 7 days evaluated. These results suggest that only when nutrients are appropriate, factors released from the brain induce the CA to synthesize enough JH to activate reproductive maturation.     

Development and physiology of follicular atresia during ovarian growth in the house fly, Musca domestica

Atretic follicles regularly occur in the ovary of the house fly, Musca domestica. The frequency of ovarian follicular atresia and the proportion of atretic follicles per ovary are related to the stage of oögenesis and to the age of the females. Only vitellogenic follicles may become atretic. The atresia may occur at any stage of vitellogenesis, though most follicles become atretic in mid-vitellogenesis. Atretic follicles are completely resorbed within 24–36 hr. The follicle cells may play a synthesizing role during growth and disintegrating one during follicle resorption. The induction of glycogen synthesis by the cessation of RNA and protein synthesis and by vitellogenesis in normal follicles is discussed. The same processes occur prematurely in the atretic follicle which can be thus distinguished by a high content of glycogen.     

Reproductive under-performance of tsetse flies in the laboratory, related to feeding frequency

Abstract. The rates of development of the eggs and larvae in utero and the next two developing ovarioles were measured by ovarian dissection on each day of the pregnancy cycle in tsetse, Glossina morsitans, subject to different feeding regimes. Compared with flies fed four times per pregnancy cycle, flies fed three times per cycle showed a lower pupal production rate (70%), the same (zero) adult mortality, a slightly slower growth rate of the larva and second ovariole only from day 8 onwards, but the same growth rate of the first ovariole. Flies fed only twice per pregnancy cycle produced no pupae, suffered 18% adult mortality and showed a significantly slower growth rate of the larva and second ovariole from days 6 and 7 respectively, but still the growth rate of the first ovariole was barely affected. Flies offered food three times or twice per pregnancy cycle engorged fully at every opportunity, but 16.5% of the flies offered food four times per cycle did not feed on every occasion, while 12–22% did not engorge fully on days 3, 5 or 7. In assessing the applicability of these laboratory results to the field situation the following points must be borne in mind: in the laboratory flies take smaller mean blood meals than in the field; during protein production associated with larval growth the proportion of the blood meal lost to transformation and excretory costs is less than during normal lipid metabolism; the balance between the tsetse's known fertility rate and adult and pupal mortality rates reveals that the abortion rate in the field must be extremely low. The high abortion rates usually observed in laboratory colonies, even when flies are offered food daily_l would be quite untenable in the field and indicate that laboratory conditions impose physiological stresses on the flies that are quite different from those in the field. These facts indicate that three field-sized meals may be sufficient to meet the energy demands of normal larval development in the field.     

Relationship between protein ingestion and sexual receptivity in females of the Australian sheep blowfly Lucilia cuprina

The receptivity of females of Luciliu cuprina to mating attempts is increased by protein feeding. Females became maximally receptive after consuming a quantity of protein which was insufficient to allow full ovarian development in any individual, and which was 25 % of that needed to produce full ovarian development in most flies. Receptivity increased progressively over 3 days following a protein meal taken on the day after emergence, but was almost maximal within 24 h when females were given a protein meal on the fifth day after emergence. Topical application of the insect growth regulator Altosid caused a marked increase in the sexual receptivity of non-protein fed females.     

Age‐related activity patterns are moderated by diet in Queensland fruit flies Bactrocera tryoni

Life‐history parameters and the fitness of tephritid flies are closely linked to diet. Studies of locomotor behaviour can provide insights to these links, although little is known about how locomotor behaviour is influenced by diet. In the present study, video recordings of Queensland fruit flies Bactrocera tryoni Froggatt (Diptera: Tephritidae) (‘Q‐flies’) that are maintained individually in cages are used to determine how diet affects the activity patterns (flight, walking, grooming, inactivity) of males and females at ages ranging from 4 to 30 days. The frequency and total duration of activities over 10‐min trials are affected by diet, age and sex. Supplementation of diet with hydrolysed yeast results in a higher frequency and duration of flight in flies of all ages and both sexes. The effect of diet on other activities varies with age. Q‐flies fed sugar only increase walking frequency steadily from 4 to 30 days post‐eclosion, whereas flies fed sugar + yeast have higher walking frequencies at 4 and 10 days than flies fed sugar only, although they then exhibit a sharp decline at 30 days post‐eclosion. The frequency and duration of inactivity remain consistent in flies fed sugar + yeast, whereas flies fed sugar only exhibit a marked increase in inactivity from 4 to 30 days post‐eclosion. Compared with older flies, 4 day‐old Q‐flies fed sugar only spend considerably more time grooming. The potential of activity monitoring as a quality control test for flies that are mass‐reared for use in sterile insect technique programmes is discussed.     

Methods of short-term storage of cultures of the blowfly <Emphasis Type="Italic">Calliphora Vicina</Emphasis> R.-D. (Diptera,Calliphoridae)

Three methods of short-term storage of the blowfly Calliphora vicina strains are considered based on the experimental study of 21 strains originating from different parts of the species range. The colony can be preserved as diapausing adults at 6° and darkness for 2–3 months or more, depending on the geographical origin of the population. During the first five days of adult life the flies should be kept at 12° and short day on a sugar diet, after which they should be transferred into a refrigerator. During artificial hibernation the flies also require periodic sugar feeding every 20 days (3–4 h at 20°C) to maintain their vital functions. The combination of temperatures of 20–23°C and a protein diet terminates reproductive diapause, and oviposition starts in 10–17 days. The fly strain may be preserved as reproductive females at 6°C and darkness with sugar feeding. Flies also require periodic sugar feeding at 20°C (3–4 hours). In this case the flies start laying eggs 2–3 days after being transferred to 20–23°C. The preservation of diapausing larvae is a more reliable method of prolonged strain storage. In this case the flies of maternal generation are maintained at 20–23°C on sugar and protein diet. The egg rafts laid during 5–6 hours are then transferred into 12°C and short day until hatchment. The hatched larvae should be immediately placed into a refrigerator (2–3 or 5–6°C), where they feed during 1–1.5 months and enter diapause. For strain restoration, the diapausing larvae should be transferred into 20–25°C, where they pupariate in 3–5 days and the flies emerge in nearly 10 days.     

Vibrio cholerae laboratory infection of the adult house fly Musca domestica

The present study was designed to test the hypothesis that house flies may be capable of specifically harbouring ingested Vibrio cholerae in their digestive tracts. Flies were continuously fed green fluorescent protein (GFP)‐labelled, non‐O1/non‐O139 environmental strains of V. cholerae. Bacterial burdens were quantitatively measured using plate counts and localization was directly observed using confocal microscopy. Vibrio cholerae were present in the fly alimentary canal after just 4 h, and reached a plateau of ～10⁷ colony‐forming units (CFU)/fly after 5 days in those flies most tolerant of the pathogen. However, individual flies were resistant to the pathogen: one or more flies were found to carry < 180 V. cholerae CFU at each time‐point examined. In flies carrying V. cholerae, the pathogen was predominantly localized to the midgut rather than the rectal space or crop. The proportion of house flies carrying V. cholerae in the midgut was dose‐dependent: the continuous ingestion of a concentrated, freshly prepared dose of V. cholerae increased the likelihood that fluorescent cells would be observed. However, V. cholerae may be a transient inhabitant of the house fly. This work represents the first demonstration that V. cholerae can inhabit the house fly midgut, and provides a platform for future studies of host, pathogen and environmental mediators of the successful colonization of this disease vector.     

Vitellin and vitellogenin in the soldier bug, Podisus maculiventris: identification with monoclonal antibodies and reproductive response to diet

A 171,000 M(r )polypeptide of Podisus maculiventris (Say) (Heteroptera: Pentatomidae) that constituted 16% of the protein in eggs also constituted up to 25% of the protein in hemolymph of fed females. It was identified as the major or sole apoprotein of vitellogenin. Eggs contained major polypeptides of 171, 106, and 51 kDa. The hemolymph polypeptide was identified with a polypeptide (vitellin) in egg extracts by comparing molecular weights, specificity of occurrence in fed females, and immunological reactivities. Females, starved for 5 days after eclosion to assure complete previtellogenic development, produced vitellogenin within a day after feeding on larval Galleria mellonella, and within 4 days after feeding on an artificial diet. Appearance of vitellogenin preceded ovarian growth by 2-3 days. Two monoclonal antibodies raised against egg proteins of P. maculiventris were selected for their strong reaction against egg extract and female hemolymph and null reaction against male hemolymph. Only one 170-kDa band in egg and hemolymph reacted with the antibodies on denaturing Western blots. These monoclonal antibodies are being used to develop an enzyme-linked immunosorbent assay (ELISA) to quantitate reproductive response of females to diets of differing quality.     

F-actin distribution in the ovaries of pre-vitellogenic and vitellogenic black blowflies,Phormia regina (Meigen) (Diptera : Calliphoridae)

The spatial distribution of F-actin microfilaments in the ovaries of previtellogenic and vitellogenic female black blowflies, Phormia regina (Diptera : Calliphoridae), as the females shift from a sugar to a liver diet, is determined using rhodamine-labelled phalloidin (rh-phalloidin). During the pre-vitellogenic stages of ovarian development (i.e. corresponding to a sugar diet) a single bright fluorescent layer marks the interface between follicle cells and the oocyte. Fluorescence is also most evident at the inner surface of the ring canals of the nurse cells. This is observed in the nurse cells both in the distal part of the germarium, and in the vitellogenic growing oocyte. However, when liver-fed (i.e. necessary for vitellogenesis), 2 bright fluorescent layers are observed at the follicle cell-oocyte interface. In addition, the cytoplasm of the nurse cells during vitellogenesis appears full of fluorescent microfilaments and the actin rings are found to increase in size and thickness. The changing organization of the F-actin microfilaments in the follicles during the process of both egg chamber and oocyte formation is discussed and possible functions considered.     

Cytodifferentiation and vitellogenesis during oogenesis in arachnida: Cytological studies on developing oocytes of a harvestman

Light and electron microscope studies were made on harvestman oocytes during the course of their origin, differentiation, and vitellogenesis. The germ cells appear to originate from the ovarian epithelium. They subsequently migrate to the outer surface of the epithelium, where they remain attached often by means of stalk cells which suspend them in the hemocoel during oogenesis. The “Balbiani bodies,” “yolk nuclei,” or “nuage” constitute a prominent feature of young, previtellogenic oocytes, and take the form of large, but variable sizes of electron-dense cytoplasmic aggregates with small fibrogranular components. The cytoplasmic aggregates fragment and disperse, and cannot be detected in vitellogenic oocytes. The young oocytes become surrounded by a vitelline envelope that appears to represent a secretory product of the oocyte. The previtellogenic oocytes are impermeable to horseradish peroxidase under both in vivo and in vitro conditions. In addition to mitochondria, dictyosomes, and abundant ribosomes, the ooplasm of the previtellogenic oocyte acquires both vesicular and lamellar forms of the rough-surfaced endoplasmic reticulum. In many areas, a dense homogeneous product appears within the cisternae of the endoplasmic reticulum and represents nascent yolk protein synthesized by the oocyte during early stages of vitellogenesis. Later in vitellogenesis, the oocyte becomes permeable to horseradish peroxidase under both in vivo and in vitro conditions. This change is associated with a massive process of micropinocytosis which is reflected in the presence of large numbers of vesicles of variable form and structure in the cortical ooplasm. Both spherical and tubular vesicles are present, as are coated and uncoated vesicles. Stages in the fusion of the vesicles with each other and with developing yolk platelets are illustrated. In the harvester oocytes, vitellogenesis is a process that involves both autosynthetic and heterosynthetic mechanisms.     

OVARIAN NUTRITIONAL RESOURCES DURING THE REPRODUCTIVE CYCLE OF THE HEMATOPHAGOUS DIPETALOGASTER MAXIMA (HEMIPTERA: REDUVIIDAE): FOCUS ON LIPID METABOLISM

In this study, we have analyzed the changes of the ovarian nutritional resources in Dipetalogaster maxima at representative days of the reproductive cycle: previtellogenesis, vitellogenesis, as well as fasting‐induced early and late atresia. As expected, the amounts of ovarian lipids, proteins, and glycogen increased significantly from previtellogenesis to vitellogenesis and then, diminished during atresia. However, lipids and protein stores found at the atretic stages were higher in comparison to those registered at previtellogenesis. Specific lipid staining of ovarian tissue sections evidenced remarkable changes in the shape, size, and distribution of lipid droplets throughout the reproductive cycle. The role of lipophorin (Lp) as a yolk protein precursor was analyzed by co‐injecting Lp‐OG (where OG is Oregon Green) and Lp‐DiI (where DiI is 1,10‐dioctadecyl‐3,3,30,30‐tetramethylindocarbocyanine) to follow the entire particle, demonstrating that both probes colocalized mainly in the yolk bodies of vitellogenic oocytes. Immunofluorescence assays also showed that Lp was associated to yolk bodies, supporting its endocytic pathway during vitellogenesis. The involvement of Lp in lipid delivery to oocytes was investigated in vivo by co‐injecting fluorescent probes to follow the fate of the entire particle (Lp‐DiI) and its lipid cargo (Lp‐Bodipy‐FA). Lp‐DiI was readily incorporated by vitellogenic oocytes and no lipoprotein uptake was observed in terminal follicles of ovaries at atretic stages. Bodipy‐FA was promptly transferred to vitellogenic oocytes and, to a much lesser extent, to previtellogenic follicles and to oocytes of ovarian tissue at atretic stages. Colocalization of Lp‐DiI and Lp‐Bodipy‐FA inside yolk bodies indicated the relevance of Lp in the buildup of lipid and protein oocyte stores during vitellogenesis.     

Characterization and expression of the putative ovarian lipoprotein receptor in the Kuruma prawn, Marsupenaeus japonicus

Ovarian low density lipoproteins (LDL) such as vitellogenin (Vg) are the precursors of the major yolk protein vitellin, and constitute the major source of nutrients serving the developing embryo. The objective of this study was to gain a better understanding of crustacean egg development by focusing on the process of Vg internalization by its receptor (ovarian LDLR). First, an ovarian LDLR cDNA sequence in Marsupenaeus japonicus was determined. Ovarian LDLR mRNA expression was then examined, and was seen to be specific to the ovary, exhibiting highest levels during the previtellogenic stage. This pattern of ovarian LDLR expression is thought to signify preparation for yolk protein incorporation into the oocyte. Using immunoblotting techniques, an ovarian LDLR band was detected whose size was similar to that estimated from the deduced amino acid sequence. The ovarian LDLR protein was expressed only at the onset of vitellogenesis, and histological studies supported these observations. This is the first occasion that the ovarian LDLR and its expression dynamics during vitellogenesis have been fully characterized in a crustacean.     

The relationship between nutrition,vitellogenin, vitellin and ovarian development in the housefly, Musca domestica L.

The effect of adult nutrition on oögenesis during the first gonotropic cycle was studied in three strains of the housefly, Musca domestica. Two of the strains were anautogenous and the third was autogenous. In these strains, three subunits (51, 43 and 42 kdaltons) of vitellogenin and vitellin were electrophoretically identical using SDS-PAGE electrophoresis for haemolymph proteins of vitellogenic females and for egg extracts. Each developmental stage of the ovary in individual females flies of both autogenous and anautogenous strains fed on either sugar or protein clearly reflected the appearance of electrophoretic bands for vitellogenin and vitellin. Using immunological analysis, a very small amount of vitellogenin was detectable in the haemolymph of previtellogenic flies. The highest level of vitellogenin appeared in the haemolymph at the middle of vitellogenic phase and reached about 25% of the total haemolymph protein. There were differences in vitellogenin concentration in females with mature eggs between the two anautogenous strains: vitellogenin was not detectable in one strain, and the other showed 30% of the maximal level.     

Effects of disodium octaborate tetrahydrate on survival, behavior, and egg viability of adult muscoid flies (Diptera: Muscidae).

Disodium octaborate tetrahydrate (Na2B8O13(.)4H2O) was mixed with sugar and fed to adult Musca domestica L. and Fannia canicularis (L.) to determine concentration-mortality relationships. LC50s (48-h exposure) were 5.7% for M. domestica and 1.0% for F. canicularis. Rates of 1 and 2% were used to test effects on M. domestica mortality and egg hatch over an 8-d period. Reduced egg hatch was evident after 1 d of feeding on the treated mixtures and was greatest (less than 10% egg hatch) after flies fed only on treated mixtures for 2 d. A partial rebound in egg hatch occurred after 3-4 d of feeding on treated diet. Sperm motility in females fed treated sugar was apparently normal. Fertile egg placed on treated poultry manure did not hatch, indicating embryonic death, which also may have been involved in the low hatch of eggs observed from treated flies. When flies were exposed to treated sugar for 2 d then returned to untreated diet, delayed mortality effects and reduced egg hatch persisted for at least 3 d. Behavioral assays (feeding) with M. domestica demonstrated that flies rejected borate-sugar mixtures in favor of sugar alone when the concentration of borate was greater than 2%. Given a choice of treated and untreated poultry manure for oviposition, flies also rejected the treated manure. The potential of borates in adult bait formulations or applied to developmental substrates for fly control is discussed.     

Seasonal variation in ovarian histology of the viviparous lizard Sceloporus torquatus torquatus

Changes in ovarian histology during the reproductive cycle of the viviparous lizard Sceloporus torquatus torquatus are described. In general, the variation in follicular histology observed during the seasonal cycle is similar to that of other lizards. Sceloporus t. torquatus exhibits a cycle in which small, previtellogenic follicles exist in the ovary from December to August. Vitellogenesis occurs between September and November, followed by ovulation from late November to early December. Parturition occurs the following spring. After ovulation, the remaining follicular cells form the corpus luteum and luteolysis did not occur until April-May. Follicular atresia is commonly observed in previtellogenic follicles with polymorphic granulosa, but occurs less frequently in follicles during late vitellogenesis. There are two germinal beds in each ovary. The yolk nucleus is evident in young oocytes as is a vacuolated ooplasma prior to vitellogenesis. Extensive polymorphism is observed in yolk platelets. Mast cells and secretory cells are observed in the thecal layer of the follicular wall as are melanocytes in the ovarian stroma. © 1995 Wiley-Liss, Inc.     

Ecdysteroid titer and oocyte growth in the northern house mosquito, Culex pipiens L

In an anautogenous strain of the northern house mosquito, Culex pipiens, the ovaries reached the resting stage (follicle length = 90 microns) three days after adult emergence. Follicle length increased from 90 to 550 microns between 0 and 60 hr after a blood meal. Total ecdysteroids reached a peak at 400 fmol/insect at 36 hr after a blood meal then declined rapidly. The ratio of 20-hydroxyecdysone to ecdysone increased in conjunction with the total ecdysteroid level. Oocyte growth beyond the resting stage and initiation of vitellogenesis was dependent on a head factor which was released within 4-8 min of the start of the blood meal.     

Regulation of the vitellogenin receptor during Drosophila melanogaster oogenesis

In many insects, development of the oocyte arrests temporarily just before vitellogenesis, the period when vitellogenins (yolk proteins) accumulate in the oocyte. Following hormonal and environmental cues, development of the oocyte resumes, and endocytosis of vitellogenins begins. An essential component of yolk uptake is the vitellogenin receptor. In this report, we describe the ovarian expression pattern and subcellular localization of the mRNA and protein encoded by the Drosophila melanogaster vitellogenin receptor gene yolkless (yl). yl RNA and protein are both expressed very early during the development of the oocyte, long before vitellogenesis begins. RNA in situ hybridization and lacZ reporter analyses show that yl RNA is synthesized by the germ line nurse cells and then transported to the oocyte. Yl protein is evenly distributed throughout the oocyte during the previtellogenic stages of oogenesis, demonstrating that the failure to take up yolk in these early stage oocyte is not due to the absence of the receptor. The transition to the vitellogenic stages is marked by the accumulation of yolk via clathrin-coated vesicles. After this transition, yolk protein receptor levels increase markedly at the cortex of the egg. Consistent with its role in yolk uptake, immunogold labeling of the receptor reveals Yl in endocytic structures at the cortex of wild-type vitellogenic oocytes. In addition, shortly after the inception of yolk uptake, we find multivesicular bodies where the yolk and receptor are distinctly partitioned. By the end of vitellogenesis, the receptor localizes predominantly to the cortex of the oocyte. However, during oogenesis in yl mutants that express full-length protein yet fail to incorporate yolk proteins, the receptor remains evenly distributed throughout the oocyte.     

Nonvitellogenic female sterile mutants and the regulation of vitellogenesis in Drosophila melanogaster.

The genetic and endocrine regulation of vitellogenesis was investigated by studying 18 female sterile mutations that disrupt the development of normal vitellogenic follicles. Applications of exogenous juvenile hormone analog and reciprocal ovarian transplants between flies of different genotypes were employed to accomplish our first two objectives: to find (1) whether the mutation blocked development of the ovary directly, and (2) whether the mutation altered the hormonal milieu. In 15 of the mutants the developmental defect was localized to the ovary, but in the other 3 the ovary was competent to respond to a permissive environment. The internal milieu of these three mutants (ap⁴, fs(3)A1, fs(2)A18) was unable to provoke normal development in wild-type ovaries, suggesting that these mutations cause endocrine defects. Our third objective was to find whether an endocrine organ was itself defective in any of these mutants. The corpus allatum from two of the mutants was unable to provoke vitellogenesis in isolated wild-type abdomens, but corpora allata from wild-type females or from other mutants were able to promote maturation of ovarian follicles in isolated abdomens. Our fourth objective was to find whether any of the mutants were able to produce yolk proteins. Immunoelectrophoresis of fly hemolymph demonstrated that in all mutants tested vitellogenins were found in the blood. These experiments permit four main conclusions. First, they identify the first Drosophila mutants in which an endocrine gland is shown to be intrinsically defective during adulthood. Second, they show that the production of morphologically normal late previtellogenic follicles is not required for the induction of vitellogenin synthesis and secretion. Third, they show that juvenile hormone can cause ovarian follicles to sequester yolk in mutant flies. And finally, they show that mutants with defective corpora allata still synthesize and secrete vitellogenin. Taken together, these conclusions suggest that in Drosophila melanogaster the uptake of vitellogenin into follicles depends upon the availability of juvenile hormone, but that the synthesis and secretion of vitellogenin are independent of both normal ovaries and totally normal corpora allata.