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The Manduca sexta Malpighian tubule assay system, developed to monitor adenylate cyclase activity, was used in combination with HPLC to isolate a novel cAMP generating peptide from 350,000 whole flesh flies, Neobellieria bullata. Mass spectrometry revealed a molecular mass of 5,047 daltons, and Edman degradation the following sequence: AGAEAEKLSGLSKYFNGTTMAGRANVAKATYAVIGLIIAYNVMKPKKK. This 48-mer peptide, called Neb-cGP, does not belong to the corticotropin releasing factor family of insect diuretic peptides. Electrophoresis and subsequent immunoblotting of peptides immunoprecipitated from a homogenate of entire flies showed that one fly contained approximately 0.003 to 0.03 micrograms Neb-cGP and that 10 micrograms represents the lowest immunostainable amount on a Western blot.
Essential vitamins for the growth of a cell line derived from the flesh fly, Sarcophaga peregrina, were determined. By examining the survivability of continuous passages of the cells in the chemically defined medium lacking one vitamin, thiamine, riboflavin, pantothenate and either niacin or niacinamide were found to be essential for the continuous growth of the flesh fly cells in vitro. [Originally published in Volume 37, Archives of Insect Biochemistry and Physiology, 37:283-286 (1998).]
The action of venom from the ectoparasitic wasp, Nasonia vitripennis, was monitored by examining alterations in patterned muscular movements characteristic of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata. Venom injected into larvae prior to pupariation caused a dose-dependent delay in pupariation. Eventually, such larvae did pupariate, but puparia were abnormally formed. Barographic records revealed that all elements of pupariation behavior were present in venom-injected larvae, but pupariation behavior was not well synchronized with tanning, thus implying that the venom caused disruption in the temporal organization of central motor programs. When larvae were ligated and injected with venom posterior to the ligature, no response was evident in the posterior region, suggesting that the venom does not directly stimulate muscles or neuromuscular junctions. Injection of exogenous ecdysteroid into venom-injected larvae restored some elements of pupariation behavior, consistent with ecdysone's role in stimulating the release of anterior retraction factor and puparium tanning factor, two factors that are released from the CNS to regulate pupariation. When the venom was injected into newly emerged imagoes, the duration of extrication behavior was shortened, whereas all phases of post-eclosion behavior were lengthened. These observations imply that the venom affects CNS centers that regulate the muscular systems engaged in extrication and post-eclosion behavior.
Two predatory fly species, Lispe consanguinea Loew, 1858 and L. tentaculata DeGeer, 1776, inhabit the supralittoral zone at the shore of a fresh-water reservoir. Both species look alike and possess similar "badges," reflective concave silvery scales on the face. Flies occupy different lek habitats. Males of the first species patrol the bare wet sand on the beach just above the surf. Males of the second species reside on the more textured heaps of algae and stones. Courtship and aggressive behaviour of males was video-recorded and analysed frame by frame. Visual stimuli provided by the conspecific partner were computed in the body-fixed space of a fly observer. Males of L. consanguinea perform long pedestrian dances of pendulating circular arcs (frequency 2 s(-1), median radius 2.5 cm, linear velocity 0.130 m/s). Right and left side runs are equally probable. Circular runs are interrupted by standby intervals of average duration 0.35 s. The female views the male as a target covering 2 by 2 ommatidia, moving abruptly with the angular velocity over 200 degrees/s in a horizontal direction down the path of about 50 degrees till the next standpoint. Dancing is evenly distributed around the female. On the contrary, the male fixates the image of the female within the narrow front sector (median +/-10 degrees); the target in his view has 6-7 times less angular velocity and angular span of oscillations, and its image in profile overlays 6-8 by 2 ommatidia. If the female walks, the male combines tracking with voluntary circular dances. Rival males circle about one another at a distance shorter than 15 mm, but not in close contact. Males of L. tentaculata are capable of similar circular courting dances, but do so rarely. Usually they try to mount any partner immediately. In the latter species, male combat consists of fierce wrestling. Flies of both species often walk sideward and observe the partner not in front but at the side.
Thoracic axons occurring in Musca and Sarcophaga are similar to those previously reported in the giant fiber pathway of Drosophila. Serial section reconstruction of both species has shown that the cervical giant fiber descending from the brain into the thoracic ganglion and the thoracic motor axon innervating the tergotrochanteral muscle follow courses matching those of similar axons in Drosophila. Likewise in both Musca and Sarcophaga a thoracic axon establishes axoaxonal synapses onto dorsal longitudinal muscle motor neurons. This axon is similar in both course and synaptic configuration to the peripherally synapsing interneuron in Drosophila. Although these similarities suggest that the three axon pairs are homologous in all three fly species, Several differences are also observed. Thus this system of identified axons may be a useful model for investigating phylogenetic variation in specific neuronal form and connectivity.
In dipterous insects, a volume of behavioral and electrophysiological studies promote the contention that three wide-field motion-sensitive tangential neurons provide a necessary and sufficient input to specific channels that drive the torque motor during flight. The present studies describe the results of neuroanatomical investigations of the relationships between motion-sensitive neuropil in the fly optic lobes and descending neurons that arise from a restricted area of the brain and supply segmental neck and flight motor neuropil. The present observations resolve at least 50 pairs of descending neurons supplying flight motor centers in the thoracic ganglia. The majority of descending neurons receive a distributed output from horizontal motion-sensitive neurons. However, the same descending neurons are also visited by numerous small-field retinotopic neurons from the lobula plate as well as hitherto undescribed small tangential neurons. Neuroanatomical studies, using cobalt, Golgi, and Texas red histology, demonstrate that these smaller inputs onto descending neurons have dendrites that are organized at specific strata in retinotopic neuropil and that these correspond to horizontal and vertical motion sensitivity layers. Conclusions that only a restricted number of wide-field neurons are necessary and sufficient for visually stabilized flight may be premature. Rather, neuroanatomical evidence suggests that descending neurons to the flight motor may each be selectively tuned to specific combinations of wide- and small-field visual cues, so providing a cooperative descending network controlling the rich repertoire of visually evoked flight behavior.
Neurons within the precursor of the adult leg, the imaginal disc, innervate a larval sense organ, Keilin's organ. Electron microscopical investigations of first instar larvae show that five dendrites end at the organ: three insert at the bases of the three hairs of the organ and two end against the cuticle, without any apparent cuticular specialization. In third instar larvae, the imaginal leg discs invaginate into the body cavity, and only four of the dendrites (the outer segments of which become greatly elongated) remain in contact with Keilin's organ. The axons of the neurons that supply Keilin's organ project into a ventral neuropile region of the central nervous system, with a pattern that resembles the projections of other larval sensilla. Electrical activity can be recorded from neurons of the imaginal disc in response to mechanical stimulation.
The imaginal leg disc of Phormia regina contains eight neurons that arise during embryogenesis. Five of the neurons are associated with Keilin's organ, and of these five, two persist to the adult fly. Two new neurons arise at about the time of pupariation and flank each of these persisting neurons, forming two triplets of cells. Both triplets can be followed throughout metamorphosis; in the late pupa they are situated anteriorly and posteriorly at the tip of the fifth tarsomere. Two triplets of cuticular specializations are found at corresponding positions in the adult fly, each consisting of two campaniform sensilla and a trichoid hair. The central member of each set of sensilla, a campaniform sensillum, is associated with the persisting cell.
In all higher dipterans (Brachycera), including the fruitfly, Drosophila melanogaster, each egg chamber (ovarian follicle) consists of a group (clone) of germ cells (one oocyte and 15 accompanying nurse cells) that is surrounded by a layer of somatic mesodermal follicular cells (FCs). As oogenesis progresses the initially uniform FCs diversify into several morphologically and functionally distinct subpopulations. In D. melanogaster some of these subpopulations, e.g., border, centripetal, and dorsolateral cells, undertake coordinated migration or rearrangement over the surface of the germ cells. During the final stages of oogenesis these subpopulations participate in the formation of a complex, regionally specialized eggshell. In representatives of lower brachycerans (Orthorrhapha), only FCs that undertake active, directed migration are the border cells. These cells originate at the anterior pole of the ovarian follicle and migrate between the nurse cells to the anterior pole of the oocyte. Reduced motility of FCs in lower brachycerans results in the absence of certain FC subpopulations in their egg chambers and subsequent simplicity of their eggshells. We found that the lack of some FC subpopulations coincided with the appearance of lamellipodium-like protrusions of the oocyte. These protrusions penetrated between the apposing membranes of nurse and FCs and partially enveloped the nurse cell compartment. Analysis of whole-mount preparations stained with rhodamine-conjugated phalloidin revealed that the protrusions contained microfilaments and that their tips were equipped with actin-rich filopodium-like processes. We also found that in some lower brachycerans (representatives of the family Rhagionidae), the FCs located at the posterior pole of the oocyte, became enlarged and morphologically similar to the anterior border cells. These findings indicate that in higher dipterans the processes leading to the formation of a functional egg are variable and often markedly different from those in the model organism, D. melanogaster.
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In the larval brain of dipteran insects, there are two medial and three lateral groups of neurons innervating the ring gland. One lateral group extends fibers to the corpus allatum. After metamorphosis, a large cluster of the medial group in the pars intercerebralis and two lateral groups in the pars lateralis innervate the retrocerebral complex and some neurons from the lateral group and a few from the medial group extend fibers to the corpus allatum in the adults. Neuropeptides such as insulin-like peptides, FMRFamide related peptides, Locusta-diuretic hormone, beta-pigment dispersing hormone, Manduca sexta-allatostatin, ovary ecdysteroidogenic hormone, and proctolin have been immunocytochemically revealed in medial groups in the pars intercerebralis, and FMRFamide related peptides, beta-pigment dispersing hormone, corazonin, and M. sexta-allatostatin in lateral groups in the pars lateralis of dipteran brains. In mosquitoes after the blood meal, ovary ecdysteroidogenic hormone from 2-3 pairs of medial neurosecretory cells is released at the corpus cardiacum to stimulate the ovaries to secrete ecdysteroid to cause ovarian development. In addition to ovarian development, removal and implantation experiments have shown that neurosecretory cells in the pars intercerebralis and pars lateralis are involved in control of reproductive diapause, cuticular tanning, sugar metabolism, and diures.
Insects that tolerate severe cold during winter may either supercool or tolerate ice forming within the tissues of the body. To compare the relative advantages of freezing and supercooling, we measured rates of CO(2) production and water loss in frozen and supercooled goldenrod gall fly larvae (Eurosta solidaginis). As an important first step, we measured the time required for ice content and metabolic rate to stabilize upon freezing. Ice content stabilized after only three hours of freezing at -5 degrees C, whereas CO(2) production required 12 hours to stabilize. Subsequent experiments found that freezing greatly reduced both water loss and metabolic rate. Comparisons of supercooled and frozen larvae at -5 degrees C indicated that CO(2) production fell 47% with freezing and water loss decreased 35%. As temperature decreased to -10 and -15 degrees C, CO(2) production fell exponentially and was no longer detectable at -20 degrees C with our measurement system. Our results demonstrate that freezing significantly reduces energy consumption during the winter and may therefore improve winter survival and spring fecundity. The advantages of freezing over supercooling would drive selection toward insect freeze tolerance and also toward higher supercooling points to increase the duration of freezing each winter.
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