Pupariation in flies: A tool for monitoring effects of drugs,venoms, and other neurotoxic compounds

A complex Sarcophaga bullata pupariation assay was used to evaluate the neurotropic effects of several drugs, venoms, and insecticides. The assay consists of tests for (1) immediate effects on the intact larva, (2) effects on ligated (ie, isolated from the central nervous system) larval abdomens, (3) morphogenetic effects on the puparium, and (4) effects on stereotyped pupariation behavior. The latter are monitored barographically by recording changes in hemocoelic pressure. Of 62 compounds screened, 18 showed morphogenetic activity at a threshold dose of 5 μg or less, 11 at a dose of 50 μg, four at a dose of 100 μg, and 29 showed no morphogenetic activity. From a comparison of the putative pharmacological actions of the tested compounds with their morphogenetic effects, certain generalizations can be made: Agents that paralyze neuromuscular systems at the peripheral level (eg, tetrodotoxin), or suppress or modify basic motor patterns centrally (eg, veratrine sulphate), cause retention of larval morphological characters in the puparium. Compounds that stimulate convulsive contractions of segmental musculature (mostly cholinergic drugs like eserine sulphate, nicotine, organophosphate insecticides) cause retention of larval segmentation on longitudinally contracted puparia. Five compounds (venom of the scorpion, Leirus quinquestriatus, pyrethrins, protoveratrine A, and kainic and quisqualic acids) stimulate musculature of the denervated abdomen. Barographic monitoring of changes in pupariation behavior appears to be a most sensitive and informative test. It reveals great differences in the ways in which compounds producing seemingly identical morphogenetic effects affect and modify behavior, thus making pharmacological classification more accurate.     

Disruption of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata Parker (Diptera: Sarcophagidae), by venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

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.     

Circulation of hemocoelic fluid during slow and fast swimming in the pteropod mollusc Clione limacina

In the pteropod mollusc Clione limacina Phipps 1774, individuals possess an open circulatory system that fills their body cavities and functions as a hydrostatic skeleton. Individuals of C. limacina demonstrate two distinct swimming behaviors, slow and fast swimming, and their wings are supported by their hydrostatic skeleton. We investigated the circulation of fluid within the body cavities of individuals of C. limacina by injecting dye into the hemocoelic compartments to visualize flow during both slow swimming and serotonin‐induced fast swimming. Hemocoelic fluid was observed to have a defined pattern of flow: rostrally from the heart into the wings and head, then following a dorsal pathway caudally into the body and tail before being taken up by the heart again. During patterned attack behavior, the neck constricted in width as the head's buccal cones were hydraulically inflated with hemocoelic fluid.     

A matrix model for studying tsetse fly populations

Some characteristics of tsetse fly population dynamics were investigated using a matrix model. To take into account the peculiarities of the tsetse fly life cycle, the classic Leslie model was modified. Our model integrated the physiological age group of Glossina females, the pupal and adult survival rate and the pupal life span. The limit of the growth rate was studied and the results were satisfactory when compared with data of tsetse fly mass rearing. The effect of adult and pupal survival rates on the growth rate was examined and confirmed the importance of adult survival. The sensitivity analysis showed that the growth rate was particularly sensitive to change in the survival rate of young nulliparous females. This matrix model, directly accessible to the experimenter, enhanced our understanding of tsetse population dynamics.     

Towards improving tsetse fly paratransgenesis: stable colonization of Glossina morsitans morsitans with genetically modified Sodalis

Background

Tsetse flies (Glossina sp.) refractory to trypanosome infection are currently being explored as potential tools to contribute in the control of human and animal African trypanosomiasis. One approach to disrupt trypanosome transmission by the tsetse fly vector involves the use of paratransgenesis, a technique that aims to reduce vector competence of disease vectors via genetic modification of their microbiota. An important prerequisite for developing paratransgenic tsetse flies is the stable repopulation of tsetse flies and their progeny with its genetically modified Sodalis symbiont without interfering with host fitness.

Results

In this study, we assessed by qPCR analysis the ability of a chromosomally GFP-tagged Sodalis (recSodalis) strain to efficiently colonize various tsetse tissues and its transmission to the next generation of offspring using different introduction approaches. When introduced in the adult stage of the fly via thoracic microinjection, recSodalis is maintained at high densities for at least 21 days. However, no vertical transmission to the offspring was observed. Oral administration of recSodalis did not lead to the colonization of either adult flies or their offspring. Finally, introduction of recSodalis via microinjection of third-instar larvae resulted in stably colonized adult tsetse flies. Moreover, the subsequent generations of offspring were also efficiently colonized with recSodalis. We show that proper colonization of the female reproductive tissues by recSodalis is an important determinant for vertical transmission.

Conclusions

Intralarval microinjection of recSodalis proves to be essential to achieve optimal colonization of flies with genetically modified Sodalis and its subsequent dissemination into the following generations of progeny. This study provides the proof-of-concept that Sodalis can be used to drive expression of exogenous transgenes in Glossina morsitans morsitans colonies representing a valuable contribution to the development of a paratransgenic tsetse fly based control strategy.

     

Identification of Tsetse (Glossina spp.) Using Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry

Glossina (G.) spp. (Diptera: Glossinidae), known as tsetse flies, are vectors of African trypanosomes that cause sleeping sickness in humans and nagana in domestic livestock. Knowledge on tsetse distribution and accurate species identification help identify potential vector intervention sites. Morphological species identification of tsetse is challenging and sometimes not accurate. The matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF MS) technique, already standardised for microbial identification, could become a standard method for tsetse fly diagnostics. Therefore, a unique spectra reference database was created for five lab-reared species of riverine-, savannah- and forest- type tsetse flies and incorporated with the commercial Biotyper 3.0 database. The standard formic acid/acetonitrile extraction of male and female whole insects and their body parts (head, thorax, abdomen, wings and legs) was used to obtain the flies'' proteins. The computed composite correlation index and cluster analysis revealed the suitability of any tsetse body part for a rapid taxonomical identification. Phyloproteomic analysis revealed that the peak patterns of G. brevipalpis differed greatly from the other tsetse. This outcome was comparable to previous theories that they might be considered as a sister group to other tsetse spp. Freshly extracted samples were found to be matched at the species level. However, sex differentiation proved to be less reliable. Similarly processed samples of the common house fly Musca domestica (Diptera: Muscidae; strain: Lei) did not yield any match with the tsetse reference database. The inclusion of additional strains of morphologically defined wild caught flies of known origin and the availability of large-scale mass spectrometry data could facilitate rapid tsetse species identification in the future.     

Visualizing the postembryonic development of Sarcophaga peregrina (flesh fly) by NMR microscopy

The postembryonic development of the flesh fly was studied using high resolution nuclear magnetic resonance imaging. Because this development occurs in a puparium, this process cannot be observed directly using standard histological techniques. The remodelling of histolysing larval tissues to developing imaginal tissues including the yellow body, a transient alimentary structure, and the integration of the developed adult structure were revealed in the images. Most surprisingly it was found that a large gas space that forms in the central region of the prepupa moves to the dorso-anterial region in less than 5 min due to the larval–pupal apolysis together with separation of the developing pupal epidermis from the puparium.     

Extrication,the primary event in eclosion,and its relationship to digging,pumping and tanning in Sarcophaga bullata

The cyclorrhaphous fly, Sarcophaga bullata (Diptera: Sarcophagidae), normally pupates within a semi-solid medium. Upon eclosion, it digs its way to an environment in which it is free of constraint and only then expands its body and wings and hardens (“tans”) its cuticle.The behaviour pattern is identical for both eclosion and digging, and is described here as a seven-phase continuum. The phases, each of which is a specific element in a series of peristaltic waves of muscular contractions and relaxations, are arranged in a deterministic sequence, for which the term “extrication behaviour” is proposed. The ability of flies to perform this sequential pattern decreases as they get older (time being measured in terms of minutes), the diminution being a depression in both the frequency and amplitude of peristaltic activity. The pattern eventually disappears at about 50 minutes of age. A study of the relationship between the onset of the post-eclosion events (i.e. expansion and hardening) which takes place after digging ends, and the decrease in intensity and, finally, termination of the extrication pattern, reveals that neither expansion nor hardening provides the feedback leading to pattern cessation.     

Less is more: restricted application of insecticide to cattle to improve the cost and efficacy of tsetse control

Studies were carried out in Zimbabwe of the responses of tsetse to cattle treated with deltamethrin applied to the parts of the body where most tsetse were shown to land. Large proportions of Glossina pallidipes Austen (Diptera: Glossinidae) landed on the belly ( approximately 25%) and legs ( approximately 70%), particularly the front legs ( approximately 50%). Substantial proportions of Glossina morsitans morsitans Westwood landed on the legs ( approximately 50%) and belly (25%), with the remainder landing on the torso, particularly the flanks ( approximately 15%). Studies were made of the knockdown rate of wild, female G. pallidipes exposed to cattle treated with a 1% pour-on or 0.005% suspension concentrate of deltamethrin applied to the (a) whole body, (b) belly and legs, (c) legs, (d) front legs, (e) middle and lower front legs, or (f) lower front legs. The restricted treatments used 20%, 10%, 5%, 2% or 1% of the active ingredient applied in the whole-body treatments. There was a marked seasonal effect on the performance of all treatments. With the whole-body treatment, the persistence period (knockdown > 50%) ranged from approximately 10 days during the hot, wet season (mean daily temperature > 30 degrees C) to approximately 20 days during the cool, dry season (< 22 degrees C). Restricting the application of insecticide reduced the seasonal persistence periods to approximately 10-15 days if only the legs and belly were treated, approximately 5-15 days if only the legs were treated and < 5 days for the more restricted treatments. The restricted application did not affect the landing distribution of tsetse or the duration of landing bouts (mean = 30 s). The results suggest that more cost-effective control of tsetse could be achieved by applying insecticide to the belly and legs of cattle at 2-week intervals, rather than using the current practice of treating the whole body of each animal at monthly intervals. This would cut the cost of insecticide by 40%, improve efficacy by 27% and reduce the threats to non-target organisms and the enzootic stability of tick-borne diseases.     

Tsetse immune system maturation requires the presence of obligate symbionts in larvae

Beneficial microbial symbionts serve important functions within their hosts, including dietary supplementation and maintenance of immune system homeostasis. Little is known about the mechanisms that enable these bacteria to induce specific host phenotypes during development and into adulthood. Here we used the tsetse fly, Glossina morsitans, and its obligate mutualist, Wigglesworthia glossinidia, to investigate the co-evolutionary adaptations that influence the development of host physiological processes. Wigglesworthia is maternally transmitted to tsetse's intrauterine larvae through milk gland secretions. We can produce flies that lack Wigglesworthia (Gmm(Wgm-) yet retain their other symbiotic microbes. Such offspring give rise to adults that exhibit a largely normal phenotype, with the exception being that they are reproductively sterile. Our results indicate that when reared under normal environmental conditions Gmm(Wgm-) adults are also immuno-compromised and highly susceptible to hemocoelic E. coli infections while age-matched wild-type individuals are refractory. Adults that lack Wigglesworthia during larval development exhibit exceptionally compromised cellular and humoral immune responses following microbial challenge, including reduced expression of genes that encode antimicrobial peptides (cecropin and attacin), hemocyte-mediated processes (thioester-containing proteins 2 and 4 and prophenoloxidase), and signal-mediating molecules (inducible nitric oxide synthase). Furthermore, Gmm(Wgm-) adults harbor a reduced population of sessile and circulating hemocytes, a phenomenon that likely results from a significant decrease in larval expression of serpent and lozenge, both of which are associated with the process of early hemocyte differentiation. Our results demonstrate that Wigglesworthia must be present during the development of immature progeny in order for the immune system to function properly in adult tsetse. This phenomenon provides evidence of yet another important physiological adaptation that further anchors the obligate symbiosis between tsetse and Wigglesworthia.     

Seasonal variation of tsetse fly species abundance and prevalence of trypanosomes in the Maasai Steppe,Tanzania

Tsetse flies, the vectors of trypanosomiasis, represent a threat to public health and economy in sub‐Saharan Africa. Despite these concerns, information on temporal and spatial dynamics of tsetse and trypanosomes remain limited and may be a reason that control strategies are less effective. The current study assessed the temporal variation of the relative abundance of tsetse fly species and trypanosome prevalence in relation to climate in the Maasai Steppe of Tanzania in 2014–2015. Tsetse flies were captured using odor‐baited Epsilon traps deployed in ten sites selected through random subsampling of the major vegetation types in the area. Fly species were identified morphologically and trypanosome species classified using PCR. The climate dataset was acquired from the African Flood and Drought Monitor repository. Three species of tsetse flies were identified: G. swynnertoni (70.8%), G. m. morsitans (23.4%), and G.pallidipes (5.8%). All species showed monthly changes in abundance with most of the flies collected in July. The relative abundance of G. m. morsitans and G. swynnertoni was negatively correlated with maximum and minimum temperature, respectively. Three trypanosome species were recorded: T. vivax (82.1%), T. brucei (8.93%), and T. congolense (3.57%). The peak of trypanosome infections in the flies was found in October and was three months after the tsetse abundance peak; prevalence was negatively correlated with tsetse abundance. A strong positive relationship was found between trypanosome prevalence and temperature. In conclusion, we find that trypanosome prevalence is dependent on fly availability, and temperature drives both tsetse fly relative abundance and trypanosome prevalence.     

The ultrastructure of the preformed ecdysial ‘line of weakness' in the puparium cap of Elenchus tenuicornis (Kirby) (Insecta: Strepsiptera).

This study describes the ultrastructure at ‘the line of weakness’ in the male puparium of Elenchus tenuicornis (Kirby) (Insecta: Strepsiptera). Superficially this line looked like an area of untanned cuticle but ultrastructurally it had an undifferentiated epicuticle, an untanned exocuticle and a loose textured endocuticle. It is speculated that the E. tenuicornis pharate adult male prior to emergence smears a chemical solution on the inner rim of the ‘line of weakness’ which dissolves the exo- and endocuticles. Following this, the slightest pressure exerted by the ptilinum of the male breaks open the cap of the puparium.     

Pollination of the orchidEpipactis thunbergii by syrphid flies (Diptera: Syrphidae)

The behavior of visitors to the flowers of the orchidEpipactis thunbergii was studied, with special attention to the role of the epichile in the pollination process. Only four species of syrphid flies legitimately pollinated the flower, among whichSphaerophoria macrogaster was regarded as the most effective pollinator. The movable epichile, possessing a furrow at its base, played a critical role in the pollination process: it threw the syrphid fly onto the stigmatic surface when both sides of the basal slanting surface of the furrow were presumably pressed in the direction of the hypochile by the fore (and middle) legs of the retreating syrphid fly. At this moment, the fly received a set of pollinia on the thorax.     

Allometry between leg and body length of insects: lack of support for the size–grain hypothesis

• 1 The size–grain hypothesis ( Kaspari & Weiser, 1999 ) states that (1) as organisms decrease in size, they perceive their environment as being more rugose; (2) long legs allow organisms to step over obstacles but hinder them from entering small gaps; and (3) as the size of an organism decreases, the benefits of long legs begin to be outweighed by the costs of construction. Natural selection should therefore favour proportionally longer legs in larger organisms, thereby leading to a positive allometry between leg and body length (scaling exponent b > 1).
• 2 Here we compare the scaling exponent of leg‐to‐body length relationships among insects that walk, walk and fly, and predominantly fly. We measured the lengths of the hind tibia, hind femur, and body length of each species.
• 3 The taxa varied considerably in the scaling exponent b. In seven out of ten groups (Formicidae, Isoptera, Carabidae, Pentatomidae, Apidae, Lepidoptera, Odonata adult), b was significantly greater than one. However, there was no gradual decrease in b from walking to walking/flying to flying insects.
• 4 The results of the present study provide no support for the size–grain hypothesis. We propose that leg length is not only affected by the rugosity of the environment, but also by (1) functional adaptations, (2) phylogeny, (3) lifestyle, (4) the type of insect development (hemimetabolism or holometabolism), and (5) constraints of gas exchange.

     

Failed predation or transportation? Causes and consequences of phoretic behavior in the pseudoscorpionDinocheirus arizonensis (Pseudoscorpionida: Chernetidae)

The pseudoscorpion Dinocheirus arizonensisinhabits rotting saguaro cactus in the Sonoran Desert and has also been found attached to the legs of the cactophilic neriid fly, Odontoloxozus longicornis.Laboratory experiments demonstrated a higher incidence of phoresy on eclosing versus postteneral adult flies, a female bias in phoresy, and an increased rate in female phoresy through time. The pseudoscorpion may also prey on the fly, but predation rate was unaffected by fly category, pseudoscorpion gender, or food deprivation. A study of pseudoscorpion colonization in the field indicated that females were the first to colonize and the first to abandon the transient habitat of a saguaro rot and, thus, corroborated patterns of phoretic behavior in the laboratory. Taken together, these results establish that phoresy is a behavior functioning specifically for dispersal. The hypothesis that pseudoscorpion transport by other arthropods is accidental, motivated by hunger, and occurs because pseudoscorpions are incapable of consuming their hosts is rejected.     

Cloning and expression of the yolk protein of the tsetse fly Glossina morsitans morsitans

Two major families of nutritional proteins exist in insects, namely the vitellogenins and the yolk proteins. While in other insects only vitellogenins are found, cyclorraphan flies only contain yolk proteins. Possible sites of yolk protein synthesis are the fat body and the follicle cells surrounding the oocyte. We report the cloning of the yolk protein of the tsetse fly Glossina morsitans morsitans, a species with adenotrophic viviparity. The tsetse fly yolk protein could be aligned with other dipteran yolk proteins and with some vertebrate lipases. In contrast to the situation in most fly species, only a single yolk protein gene was found in the tsetse fly. Northern blot analysis showed that only the ovarian follicle cells, and not the fat body represents the site of yolk protein synthesis.     

Ultrastructural localization of unique neurosecretory granules in the corpora cardiaca of the stable fly,Stomoxys calcitrans,and the Tsetse Fly,Glossina morsitans

Ultrastructural analysis of the corpora cardiaca of the stable fly, Stomoxys calcitrans, and the tsetse fly, Glossina morsitans, revealed the presence of elementary neurosecretory granules (ENG) unique to the intrinsic neurosecretory cells (INC) of these species. In addition to electron‐dense spheres, the INC of the corpus cardiacum of the stable fly contain electron‐dense angular granules, either square or rectangular in shape, while the INC of the tsetse fly contain electron‐dense spindle‐shaped ENG. The distinctive granules of these INC can be traced within nerves to their sites of storage and release, eliminating the need for labeling with artificial probes. Although the INC of the corpus cardiacum of most species have been found to be fuchsinophilic, neither the INC of the stable fly nor the tsetse fly are aldehyde‐fuchsinophilic. These peptigenic cells offer neuroendocrinologists a unique opportunity to study the physiology and biochemistry of neurosecretory cells. J. Morphol. 240:155–168, 1999. Published 1999 Wiley‐Liss, Inc.