Eosinophil and mast cell expression in host skin during larval development of the human bot fly Dermatobia hominis

Eosinophils and mast cells in the skin of Wistar rats (Rattus norvegicus) infested with Dermatobia hominis larvae were quantified and analysed. Eosinophils in parasitised skin increased markedly until 10 days post-infestation (dpi) and then decreased up to 28 dpi, close to the point at which third stage larvae (L3) emerged from the host. In ascending order, the highest numbers of eosinophils were seen in rats at 1, 4, 28, 20, 15 and 10 dpi, corresponding to the first, (1 and 4) third (20 and 28) and second (10 and 15) instars. Except for 1 dpi, eosinophil numbers were significantly higher than those seen in control animals. Mast cell numbers were highest in early infestation (4 dpi), followed by those at 20 dpi. In increasing order, numbers of mast cells were greatest at 10, 28, 15, 1, 20 and 4 dpi, although significant differences with control animals were only seen at 10 and 28 dpi. Eosinophils and mast cells showed negative correlation only in animals with second instar larvae (10 and 15 dpi). Comparative analyses were also carried out after considering the skin into four distinct regions. The results suggest that the expression of both cell types, particularly eosinophils, is an important host response to infestation by D. hominis.     

Experimental skin lesions from larvae of the bot fly Dermatobia hominis

Skin biopsies from larvae of Rattus norvegicus, experimentally infested with Dermatobia hominis (Linnaeus Jr) (Diptera: Cuterebridae), were processed for histopathological studies. Two days after infestation, the first-stage larvae (L1) were located deep in the dermis, surrounded by an inflamed area infiltrated predominantly by neutrophils. On the fourth day a thin necrotic layer could be seen close to the larvae, surrounded by large numbers of neutrophils, lymphocytes, macrophages with a few eosinophils and mast cells. A small warble was formed after the fourth day, increasing in size until the seventh day, when the L1 moulted to the second-stage larva (L2). The inflammatory process continued with increasing numbers of neutrophils, macrophages, lymphocytes, eosinophils and mast cells invading the area, as well as the proliferation of fibroblasts and endothelial cells and the appearance of a few localized haemorrhages. After 18-20 days, the L2 moulted to the third-stage larva (L3), when a few plasma cells could be seen in the inflamed area. At 25-30 days there was a reduction in the necrotic layer, as well as in the number of neutrophils and lymphocytes, although large amounts of eosinophils, plasma cells, and collagen fibres were seen. The L3 usually left the host after 30 days. Two days later, the larval cavity was reduced, mast cells infiltrated the region and collagen fibre production were increased. After 7 days, an intense infiltration of plasma cells and scattered necrotic areas could be seen. A scar formed after 10 days. This study showed the laboratory rat to be a suitable model for studies of D. hominis infestation.     

Mapping the larval midgut lumen proteome of Helicoverpa armigera, a generalist herbivorous insect

The gut lumen is the primary site of digestion and detoxification and thus presents conditions hostile to most proteins. We used 2D-gel electrophoresis and MS/MS de novo peptide sequencing to identify the major proteins stable enough to persist in the midgut lumen of caterpillars of the cotton bollworm Helicoverpa armigera, a generalist herbivorous insect and a major crop pest worldwide. As expected, we found several enzymes responsible for digestion of carbohydrates, proteins, and lipids. In addition, we identified nondigestive proteins such as a multidomain lipocalin, a protein with pathogen recognition domains, an arginine kinase related to a class of major human allergens, and abundant proteins of unknown function. Identification of the set of proteins that are secreted into the lumen will enable us to further characterize the nutritional and defensive functions of this important intraorganismal space.     

Inflammatory reaction to the human bot-fly,Dermatobia hominis,in infested and reinfested mice

Two groups of mice were infested with first stage larvae of the human bot-fly, Dermatobia hominis (Linnaeus Jr) (Diptera: Oestridae). In the first group, skin biopsies were carried out 1, 3, 5, 7, 10 and 18 days after infestation. The second group was also infested but had all the larvae removed 5 days after infestation. The mice in the latter group were reinfested 4 weeks later and skin biopsies were carried out 1, 3, 5, 7, 10 and 18 days after reinfestation. In the first group, an inflammatory reaction began slowly, the neutrophils being the main inflammatory cells, eosinophils being scarce. The reaction progressed with time, developing a necrotic halo around the larvae containing inflammatory cells surrounded by fibroblasts. The inflammation invaded the adjacent tissue. In the second group, the inflammatory reaction was intense on the day immediately after reinfestation, the pattern being changed by the presence of a large number of eosinophils. Activated fibroblasts surrounding the necrotic area around the larvae appeared 3 days after reinfestation in the second group and 7 days after infestation in the first group. The results demonstrated that the previous contact with the antigens elicited the early arrival of eosinophils, probably through the chemotactic factors liberated by mast cells in the anaphylactic reaction.     

Scanning electron microscopy studies on the first-instar larva of Dermatobia hominis

Abstract. First-instar larvae of Dermatobia hominis collected 1, 4 and 7 days after having penetrated experimentally infected rats, were studied by scanning electron microscope (SEM) observation. On the pseudocephalon there are basiconic and trichoid sensilla (antennal sensory complex), and basiconic, coeloconic and campaniform sensilla (maxillary sensory complex). The thoracic segments bear several rows of small, backwardly pointed, spines, and trichoid, campaniform, coeloconic and pit sensilla. The anterior spiracle is a minute opening. Both small and large spines directed posteriorly are on the first to fourth abdominal segments, which also bear coeloconic and companiform sensilla. These sensilla are present on the unarmed (fifth and sixth) and armed (seventh) abdominal segments. The seventh and the last (eight) abdominal segments have forwardly directed spines. Each spiracular plate has two spiracular openings and four spatulate-like structures called sun rays. The anus and the coeloconic sensilla are proeminent on the last segment. The results are compared with other parasitic dipteran larvae, and emphasize that the multiple types of sensilla on D. hominis larva may have importance in establishing the parasitic phase of the life cycle of this insect.     

Ultrastructure of the ovary of Dermatobia hominis (Diptera: Cuterebridae). I. Development during the 3rd larval instar

The ultrastructure and distribution of gonial and somatic cells in the ovary of Dermatobia hominis was studied during the 3rd larval instar. In larvae weighing between 400 and 500 mg, the ovary is partially divided into basal and apical regions by oblong somatic cells that penetrate from the periphery; these cells show ovoid nucleus and cytoplasm full of microtubules. In both regions, gonial cells with regular outlines, large nucleus and low electron-density cytoplasm are scattered among the interstitial somatic cells. These later cells have small nucleus and electrondense cytoplasm. Clear somatic cells with small nucleus and cytoplasm of very low electron-density are restrict to the apical region of the gonad. Degenerating interstitial somatic cells are seen in the basal portion close to the ovary peduncle. During all this larval period the morphological features of the ovary remain almost the same. At the end of the period there is a gradual deposition of glycogen in the cytoplasm of the somatic cells, increase in the number and density of their mitochondria plus nuclear modification as membrane wrinkling and chromatin condensation in masses.     

Testicular maturation in the sheep bot fly Oestrus ovis

The process of testicular maturation in relation to intrapuparial development was studied in the sheep nasal bot fly, Oestrus ovis L. (Diptera: Oestridae). After formation of the puparium during larval-pupal apolysis and the cryptocephalic pupal stage (approximately 24-72 h), spermatogonia had undergone mitotic divisions and sperm cysts had been formed. Five days after pupariation, spermatogonia transformed into primary spermatocytes during the phanerocephalic pupal stage, and secondary spermatocytes first appeared during the pupal-adult apolysis. Secondary spermatocytes began undergoing the second meiotic division by day 8 (transparent-eye pharate adult stage). By days 9 and 10, round spermatids were present and began to elongate by day 11. By day 12, the first bundles of tailed spermatozoa had appeared. By day 15 (the yellow-orange eye pharate adult stage), round, elongated, tailed and bundled spermatids were predominant and by day 17 differentiating spermatids occupied nearly 35% of the testicular cavity, and 60% was occupied by free sperm. By day 21 (the red-brown eye pharate adult stage), spermatozoa colonized the seminal vesicle. At emergence (approximately day 22), a complement of free sperm occupied the testis and the seminal vesicle, but groups of developing cells frequently remained in certain zones. Spermatogenesis was carried out after pupariation and spermiogenesis occurred during the pharate adult stage. After emergence, males possessed fully formed spermatozoa ready for ejaculation.     

Fine structure of the larval midgut of the fly Rhynchosciara and its physiological implications

The midgut of Rhynchosciara americana larvae consists of a cylindrical ventriculus from which protrudes two gastric caeca formed by polyhedral cells with microvilli covering their apical faces. The basal plasma membrane of these cells is infolded and displays associated mitochondria which are, nevertheless, more conspicuous in the apical cytoplasm. The anterior ventricular cells possess elaborate mitochondria-associated basal plasma membrane infoldings extending almost to the tips of the cells, and small microvilli disposed in the cell apexes. Distal posterior ventricular cells with long apical microvilli are grouped into major epithelial foldings forming multicellular crypts. In these cells the majority of the mitochondria are dispersed in the apical cytoplasm, minor amounts being associated with moderately-developed basal plasma membrane infoldings. The proximal posterior ventriculus represents a transition region between the anterior ventriculus and the distal posterior ventriculus. The resemblance between the gastric caeca and distal posterior ventricular cells is stressed by the finding that their microvilli preparations display similar alkaline phosphatase-specific activities. The results lend support to the proposal, based mainly on previous data on enzyme excretion rates, that the endo-ectoperitrophic circulation of digestive enzymes is a consequence of fluid fluxes caused by the transport of water into the first two thirds of midgut lumen, and its transference back to the haemolymph in the gastric caeca and in the distal posterior ventriculus.     

Digestion of host immunoglobulin and activity of midgut proteases in the buffalo fly Haematobia irritans exigua

The digestion of blood by the buffalo fly (Haematobia irritans exigua) was monitored for 6h at 33 degrees C after a single meal. Following the meal, the concentration of soluble protein within the midgut increased to a peak at 2 hours then decreased steadily over the next 4h. The magnitude of the increase in soluble protein at 2h indicated a release of protein from another source; most likely from lysed red blood cells. The immunoglobulin (IgG) fraction of the blood meal was digested rapidly (50% within one hour of feeding) and fully digested within 4h. This is indicative of its accessibility to digestive enzymes within the midgut. In contrast, when flies had continuous access to blood, the concentration of IgG in the midgut remained at a more constant level. The loss of antigen-binding activity of a specific antibody was more rapid than complete degradation of the IgG, with 70% of binding activity lost within one hour of feeding. The level of trypsin activity in the midgut increased from pre-feeding levels to reach a peak at 2h before returning to basal levels after 6h. The pattern of trypsin activity follows closely that of the concentration of soluble protein in the midgut (r=0.88). The activity of leucine aminopeptidase in the midgut also increased immediately after feeding and remained elevated for 4 h before declining to a basal level after 6h. The rapid digestion of IgG and subsequent loss of antibody activity suggests that for a specific anti-buffalo fly antibody to be effective it would need to be able to either evade the digestive system or induce a rapid response.     

Development of polymorphic microsatellite markers for the human botfly, Dermatobia hominis (Diptera: Oestridae)

In this report, we describe the development of 17 polymorphic microsatellite markers for the human botfly, Dermatobia hominis, an obligatory parasite of mammals of great veterinary importance in Latin America. The number of alleles ranged from 5 to 21 per locus, with a mean of 12.2 alleles per locus. The expected heterozygosity ranged from 0.2571 to 0.9206 and from 0.2984 to 0.9291 in two populations from Brazil. These markers should provide a high resolution tool for assessment of the fine-scale genetic structure of natural populations of the human botfly.     

两种侵袭宿主方式的优势种马胃蝇飞行能力研究

为了解两种不同侵染宿主方式马胃蝇的飞行行为,本研究利用飞行磨系统测定了黑腹胃蝇Gasterophilus pecorum（以牧草为产卵载体）和肠胃蝇G. intestinalis（以宿主体毛为产卵载体）的飞行能力。结果表明:（1）肠胃蝇总飞行时间和距离均显著高于黑腹胃蝇,分别为后者的5.52倍和7.65倍,但平均飞行速度无显著性差异（P>0.05）。（2）黑腹胃蝇雌虫的飞行时间、距离和速度均略高于雄虫,而肠胃蝇雌虫除平均飞行速度外的飞行参数均低于雄虫。（3）肠胃蝇吊飞期间的体重消耗（24.38%）显著高于黑腹胃蝇（14.07%）;黑腹胃蝇雌雄成虫甘油三酯含量均显著下降,但两者差异不显著（P>0.05）。飞行距离差异反映了两种不同侵染宿主方式马胃蝇的飞行能力发生了适应性的变化,而总飞行时间为两种马胃蝇飞行距离差异的主导因素。     

Structure of the larval midgut of the fly Chironomus thummi and its relationship to sites of cadmium sequestration

The midgut structure of 4-7 day old fourth instar Chironomus thummi larvae was investigated with the light arid electron microscopes. Four regions are present which may be identified by the following major features: (1) Anterior I: this is the region under the esophageal invagination. (2) Anterior II: short microviili characterize these cells. Long narrow, basal plasma membrane infoldings associated with mitochondria are conspicuous. This region is hypothesized to be important in ion and fluid transport. (3) Anterior III: numerous crystals are seen in these cells. Storage is proposed to be a major function of this region. (4) Posterior: these cells have long microviili, extensive RER, many Golgi, and short basal plasma membrane infoldings. Posterior cells probably function in secretion of digestive enzymes and absorption of nutrients. Cadmium is sequestered by the midgut, almost exclusively in the posterior cells. This phenomenon is discussed.     

Energetic cost of bot fly parasitism in free-ranging eastern chipmunks

The energy and nutrient demands of parasites on their hosts are frequently invoked as an explanation for negative impacts of parasitism on host survival and reproductive success. Although cuterebrid bot flies are among the physically largest and most-studied insect parasites of mammals, the only study conducted on metabolic consequences of bot fly parasitism revealed a surprisingly small effect of bot flies on host metabolism. Here we test the prediction that bot fly parasitism increases the resting metabolic rate (RMR) of free-ranging eastern chipmunks (Tamias striatus), particularly in juveniles who have not previously encountered parasites and have to allocate energy to growth. We found no effect of bot fly parasitism on adults. In juveniles, however, we found that RMR strongly increased with the number of bot fly larvae hosted. For a subset of 12 juveniles during a year where parasite prevalence was particularly high, we also compared the RMR before versus during the peak of bot fly prevalence, allowing each individual to act as its own control. Each bot fly larva resulted in a ~7.6% increase in the RMR of its host while reducing juvenile growth rates. Finally, bot fly parasitism at the juvenile stage was positively correlated with adult stage RMR, suggesting persistent effects of bot flies on RMR. This study is the first to show an important effect of bot fly parasitism on the metabolism and growth of a wild mammal. Our work highlights the importance of studying cost of parasitism over multiple years in natural settings, as negative effects on hosts are more likely to emerge in periods of high energetic demand (e.g. growing juveniles) and/or in harsh environmental conditions (e.g. low food availability).     

Morphology of the second- and third-instar larvae of Dermatobia hominis by scanning electron microscopy

Larvae of Dermatobia hominis 10–27 days old were collected from experimentally infected rats and their morphology was studied by scanning electron microscopy. The moult from the second to third instar occurs at 18 days, with emergence from the host at 30 days post-infection. The second-instar larvae bear on the pseudocephalon, antennae (coeloconic sensilla), and coeloconic and basicoconic sensilla on the maxillary sensory complex. The thoracic segments bear small backwardly-directed spines anteriorly and ventral trichoid and campaniform sensilla. The first four abdominal segments have small and large backwardly-directed spines that are absent on segments five and six. The seventh and eighth abdominal segments have medium-sized forwardly-directed spines. Abdominal segments are encircled by campaniform sensilla. The terminal end of the eighth abdominal segment bears the anus, prominent anal lobes and two spiracular openings on each spiracular plate. Spiracular plates show a radial sun ray pattern. The rear abdomen also bears an ecdysal aperture, several pores and eight coeloconic sensilla. Although there are slight morphological differences, the spines (predominantly flat and thorn-like) and sensilla (campaniform and coeloconic) of the third-instar larvae show a similar arrangement to that of second-instar larvae. Thoracic trichoid sensilla are not seen in third-instar larvae. A perispiracular gland aperture is situated above each posterior spiracular opening. These morphological features are compared with those of other cuterebrid larvae.     

Micro-organisms in the midgut of tsetse fly larvae.

Two types of micro-organisms were found in the midgut of Glossina morsitans larvae, a large Gram-negative bacterial rod and a small Gram-negative rickettsia-like micro-organism, although the occurrence of the rickettsial type is restricted. The location of these micro-organisms in a small area of the proventriculus of all three larval instars is discussed. The large micro-organisms resemble milk-gland bacteria, and further evidence is presented in support of a milk transmission hypothesis for these micro-organisms.