Arrested development of the rabbit stomach worm Obeliscoides cuniculi: resumption of development of arrested larvae throughout the course of a single infection

Arrested development of the rabbit stomach worm Obeliscoides cuniculi: resumption of development of arrested larvae throughout the course of a single infection. International Journal for Parasitology 16: 47–54. Twenty rabbits were inoculated once with cold treated L3s of Obeliscoides cuniculi with a high propensity for arrest and killed in pairs at intervals over 234 days post-infection. An exponential decline in numbers of adult worms and arrested larvae was observed. Arrested larvae resumed development at a rate seven times greater than adults were lost and appeared to replenish the adult population. More male than female arrested larvae were intially established in rabbits but males resumed development at a greater rate than females. Egg and larval production were correlated with the number of female worms present at any one time. In a subsidiary study, arrested larvae were recovered from one rabbit and transferred sequentially through two further rabbits. These larvae resumed development at the same rate as larvae in the 20 rabbits. Both studies suggest that resumption of development is an intrinsic characteristic of the larvae themselves and may be genetically controlled.     

Nematospiroides dubius: arrested development of larvae in immune mice.

When immune NIH mice were killed 10 days after a challenge infection with Nematospiroides dubius, approximately 10% of the inoculated larvae were recovered from the intestinal lumen, irrespective of the dose administered. When such mice were treated with cortisone from Day 10 for a period of 8 to 14 days and were subsequently killed for worm counts, it was found that they had significantly more worms than the immune control mice killed on Day 10. During the week following the beginning of treatment with cortisone there was little change in the low worm burdens in immune mice. However, 9 to 11 days after this treatment worm counts indicated that worms were accumulating in the intestinal lumen, and concurrently eggs were recorded in the feces of the mice. These observations indicated that a period of 9 to 11 days was required after the initiation of cortisone treatment on Day 10 for the worms in immune mice to complete their development to the adult lumen-dwelling stage. It is suggested that the larvae in the challenge infection became arrested early in their development in the intestinal wall and that growth resumed only after cortisone treatment. When treatment with cortisone was initiated later after challenge, it was still effective in reactivating arrested worms, but the lower worm recoveries in these mice indicated that the arrested larvae were being slowly rejected by the host. In subsequent experiments it was established that the arrested larvae of N. dubius were insusceptible to the activity of pyrantel embonate, an anthelmintic which is 99% effective against adult worms in the intestinal lumen. The mechanism whereby the larvae of N. dubius became arrested in immune mice and subsequently resumed their development after cortisone treatment is discussed.     

Methylxanthine inhibition of neurosecretion in the brain and corpus cardiacum of Cecropia

The fine-structure of the median neurosecretory cells and corpora cardiaca of the Cecropia silkmoth during the first 7 days after transfer from cold conditions to room temperature was compared to that of similar animals whose development was arrested with aminophylline. The major difference observed was the failure of the intrinsic secretory cells of the corpus cardiacum to degenerate in the arrested animals. This failure to degenerate coincides with the expected period of brain hormone release. After long periods of arrest, the medial neurosecretory cells and their axons became distended with neurosecretory granules. The significance of these observations in the initiation of adult development is discussed.     

Schistogram changes after administration of antischistosomal drugs in mice at the early phase of Schistosoma mansoni infection

Mice infected with Schistosoma mansoni were treated with oxamniquine, praziquantel, artesunate at the pre-patent phase, aiming at observing schistogram alterations. Half of the animals were perfused five days post-treatment for counting and classification of immature worms, based on pre-established morphological criteria (schistogram); the remaining animals were evaluated 42 or 100 days after infection and perfusion of the portal-system was performed for collection and counting of adult worms and oogram. It was observed that oxamniquine and artesunate treatment administered at the pre-postural phase causes significant reduction in the number of immature and adult worms. However, there was little reduction with praziquantel when used at the dose of 400 mg/kg for treatments administered 14, 15, 21 or 23 days post-infection. Artesunate was responsible for significant alterations in development of young worms, as well as for a higher number of worms presenting intestinal damages. Immature adult worms were detected in mice treated with artesunate or oxamniquine at the pre-patent phase of infection and recovered by perfusion 100 days after infection. Schistogram proved to be a very useful tool for experimental evaluation of the activity of antischistosomal drugs and a good model to identify the most sensitive stages to drugs.     

The effect of digging on development of adult characters in blowflies Calliphora erythrocephala

Digging delays expansion after emergence of adult Calliphora. If flies are kept digging for 12 to 14 hr they lose the capacity to expand and, if they are then reared for 10 days, do not develop the normal adult corpus allatum, ovaries, and cuticle. In particular, ultrastructural examination of the resilin tendon of the pleurotergal muscle shows that development is arrested at a stage similar to that in the late pharate adult but the resilin is not cross-linked. It is suggested that bursicon release is irreversibly inhibited in flies that fail to expand normally.     

Larval development of Diploptera punctata reared alone and in groups

Larvae of the cockroach Diploptera punctata were reared in isolation, in pairs, or in groups of 8–10. Duration of larval development, age at each ecdysis, weights at birth and ecdyses, and adult head-capsule width were measured. Duration of larval development was longer and adult size was larger in isolated animals than in animals reared in pairs and groups. The effect of isolation on development was more pronounced in males. All females had 4 larval instars, whereas males had 3 or 4 instars. The proportion of males with 4 larval instars was higher among animals reared in isolation. There was no difference in the duration of larval development or adult size between pair- and group-reared animals. The sex of animals in the group did not affect adult size or the duration of larval development. Males which underwent 3 or 4 larval instars had different schedules of moulting. Rates of growth of males of both instar types reared in isolation and pairs were similar. Greater adult weight of isolated animals and 4-instar-type males was a result of their longer duration of larval development. Both a higher rate of growth and longer duration of larval development contribute to the larger adult size of females than males.     

The effects of cross-fostering on the behaviour of two species of North American lemmings, Dicrostonyx groenlandicus and Lemmus trimucronatus: I. Olfactory preferences

To determine the role of the maternal environment in the development of adult olfactory preferences, the young of two species of North American lemmings (Dicrostonyx groenlandicus and Lemmus trimucronatus) were reciprocally cross-fostered shortly after birth. In-fostered (reared by foster conspecific females) and non-fostered animals served as controls. When adult, subjects were tested for olfactory preferences in a free-choice Y-maze olfactometer. When compared to controls, cross-fostered animals showed increased preference for heterospecific odour. This effect was stronger for males than for females. These results suggest that adult species-specific odour preferences are learned during the neonatal period.     

Ancylostoma caninum: Adult worm removal,corticosteroid treatment,and resumed development of arrested larvae in dogs

Beagles, 2 months old and helminth naive, were infected with chilled arrest-prone larvae of Ancylostoma caninum. Eighteen days after infection, the pups were treated with an adulticidal anthelmintic (disophenol or dichlorvos) to remove adult worms while allowing dormant larvae to survive. Examinations of treated pups at necropsy demonstrated that the remaining hypobiotic larvae could develop and mature in the same dogs within which their development was arrested. Removal of adult worms was not a stimulus for resumption of larval development. Indeed, larvae resumed development in untreated control dogs harboring substantial populations of adult worms. Prednisolone treatment of dogs, although apparently producing some degree of immunodepression as judged by lymphocyte transformation assays, did not release larvae from dormancy. In fact, the dogs treated with the corticosteroid harbored significantly greater populations of hypobiotic larvae at 100 days after infection than did their untreated controls. Some hypobiotic larvae appeared to resume development spontaneously and idiosyncratically during the 2 to 3 month duration of these experiments. Whether a synchronous resumption of development would occur given other stimuli or spontaneously after a longer period of dormancy remains to be determined.     

Arrest of adult development in debrained pupae of the silkworm, Bombyx mori

The occurrence of developmental arrest after brain removal in pupae of Bombyx mori was examined using a racial hybrid Chinese No. 115 × Japanese No. 122. The results are as follows: (1) Adult development was blocked for a long period in most insects debrained shortly after larval-pupal ecdysis; (2) the earlier the brain removal, the more arrested pupae were obtained; (3) the critical period of brain hormone secretion for adult development was earlier in the female than in the male; (4) the developmental arrest which had been induced in female pupae tended to terminate earlier than that in males; (5) the developmental arrest which was induced by extremely early brain removal terminated earlier than that obtained by later brain removal.     

Pharmacological manipulation of gastrocnemius muscle blood flow in an animal model of reperfusion injury

Despite technically satisfactory surgery for acute lower limb ischaemia reperfusion injury may result in failure of limb salvage and the need for amputation. An animal model using the rat hind limb has been developed which demonstrates this complication. A tourniquet was applied to one hind limb for 6 h and then released. Gastrocnemius muscle blood flow in both hind limbs was assessed using radiolabelled microspheres and a perfusion index calculated between the revascularized and normal hind limbs and the results compared with similar measurements in control animals and rats with a tourniquet still in situ (ischaemic). Following 10 min the median perfusion index in reperfused animals was significantly less than that in control animals (0.12 ± 2 inter-quartile range 0.02–0.43) versus 1.05 (0.68–1.18), P < 0.01) but similar to the results in rats with a tourniquet still in situ [0.04 (0.00–0.07), ns], thus demonstrating low reflow following tourniquet release. After 120 min revascularization a phase of relative reperfusion occurred with perfusion indices becoming higher than those in animals with a tourniquet in situ (0.48 (0.11–0.70) versus 0.02 (0.01–0.07), P < 0.05) but remaining lower than those in control rats [0.97 (0.79–1.13), P < 0.05]. Finally after 240 min, reperfusion injury occurred with perfusion being similar to that in animals with a tourniquet applied [0.05 [0.01–0.38) versus 0.03 (0.00–0.07), ns] and less than that in the normal rats [1.01 (0.73–1.16), P < 0.01]. Attempts to modify these reductions in muscle perfusion pharmacologically have demonstrated that a superoxide radical scavenger (superoxide dismutase and catalase) abolished low reflow (0.94 (0.54–1.12) versus 0.12 (0.02–0.43), P < 0.01). In contrast dimethylthiourea, an hydroxyl radical scavenger, prevented the phase of relative reperfusion (0.04 (0.02–0.21) versus 0.48 (0.11–0.70), P < 0.01. Sodium nitroprusside, a vasodilator, increased perfusion at 240 min [0.41 (0.11–1.22] versus 0.05 (0.01–0.38], P < 0.05] with 6 out of 15 rats having a normal perfusion index at the time when reperfusion injury would be expected to occur.     

Ancylostoma duodenale: maintenance through six generations in helminth-native pups.

An Indian strain of Ancylostoma duodenale with a known propensity for arrested development in man was passaged serially in 2-month-old, helminth-naive, male beagles. The sixth passage was initiated about 500 days after laboratory maintenance began. Experimental animals were infected by stomach tube with 1500 larvae each. These infections became patent in 22–31 days. In a line passaged in dogs without the use of a corticosteroid (prednisolone), adaptation, as judged by fecal egg count, increased through the first three generations and then declined sharply. This line appears destined for extinction. In another line passaged in prednisolone-treated pups, the strain's original viability is being maintained. Infections were long lived, frequently surviving for 90 days or more. Maximum egg output in terms of eggs per gram of feces (EPG) was highly variable between generations and between individuals within generations, but commonly exceeded 1500 EPG. In pups necropsied between 44 and 55 days, harboring fifth- and sixth-generation infections, adult worm burdens ranged from 47–146 and 34–128 in the nonprednisolone and prednisolone lines, respectively. Arrested larvae have been found in the same pups and apparently contribute to a turnover in the population of adult worms. Although A. duodenale has yet to be fully adapted to dogs in the sense that indefinite survival without prednisolone is assured, the present system yields a variety of life history stages in quantities suitable for many experimental purposes. Thus it provides a utilitarian laboratory model for the investigation of one of man's major hookworms.     

Bursicon in the mealworm, Tenebrio molitor L. and its role in the control of postecdysial tanning

Using the adult Calliphora bioassay, we found that the tanning hormone, bursicon, is present in the blood of pupal and adult Tenebrio only at the time of ecdysis, when it is released massively from the thoracic and abdominal central nervous system. The hormone's half life in the blood is short (about 1–2 h). Contrary to the findings of other workers, we could find no evidence for the presence of the hormone in the haemolymph during pharate adult development, before ecdysis begins. When newly ecdysed pupae were ligated about the neck, adult development of the thorax and abdomen proceeded normally, but postecdysial tanning of the adult cuticle was almost completely prevented. This failure to tan was not due to lack of bursicon as the hormone was released normally in the ligated animals at the time of ecdysis. This suggests that a pre-ecdysial signal may be required for the development of epidermal competence to respond to bursicon.     

Nerve growth factor-mediated induction of tyrosine hydroxylase and of neurite outgrowth in cultures of bovine adrenal chromaffin cells: dependence on developmental stage

Adrenal medullary cells were cultured in a serum-free medium from fetal, neonatal (calves), and adult bovine animals. Neurite outgrowth in response to nerve growth factor (NGF) was observed in cells obtained from fetuses up to a gestational age of 3 months but not in cultures from older animals. The tyrosine hydroxylase (TH) specific activity was found to depend on the cell density and corresponded, at a density of 2 × 10⁵ cells/cm², to the specific activity found in vivo. The TH specific activity increased about sevenfold from fetuses to adult animals. Administration of NGF in vitro caused an increase of the TH specific activity in fetal cells by up to 140% and in calf cells typically by 70–100%. Cultures from adult animals showed no significant TH increase in response to NGF. Scatchard analysis and kinetic studies of the NGF binding at 0°C to intact adrenal medullary cells cultured from calves or from adult bovine animals revealed the presence of only one class of receptors, having a dissociation constant (K_D) of 1 × 10 ⁹, M. There are 16,000 binding sites per cell. The affinity of the reeptors in vivo (determined in crude membrane preparations) did not alter during development, whereas the receptor density decreased with increasing fetal age, but was the same for calves and adults. Whereas the loss of NGF-mediated fiber outgrowth during development might be related to the reduction of receptor density, the disappearance of the NGF-mediated TH induction does not correlate with changes in the binding characteristics of NGF to the adrenal chromaffin cells.     

Elimination of Schistosoma mansoni Adult Worms by Rhesus Macaques: Basis for a Therapeutic Vaccine?

Background

Among animal models of schistosomiasis, the rhesus macaque is unique in that an infection establishes but egg excretion rapidly diminishes, potentially due to loss of adult worms from the portal system via shunts or death by immune attack.

Principal Findings

To investigate this, six rhesus macaques were exposed to Schistosoma mansoni cercariae and the infection monitored until portal perfusion at 18 weeks. Despite a wide variation in worm numbers recovered, fecal egg output and circulating antigen levels indicated that a substantial population had established in all animals. Half the macaques had portal hypertension but only one had portacaval shunts, ruling out translocation to the lungs as the reason for loss of adult burden. Many worms had a shrunken and pallid appearance, with degenerative changes in intestines and reproductive organs. Tegument, gut epithelia and muscles appeared cytologically intact but the parenchyma was virtually devoid of content. An early and intense IgG production correlated with low worm burden at perfusion, and blood-feeding worms cultured in the presence of serum from these animals had stunted growth. Using immunoproteomics, gut digestive enzymes, tegument surface hydrolases and antioxidant enzymes were identified as targets of IgG in the high responder animals.

Significance

It appears that worms starve to death after cessation of blood feeding, as a result of antibody-mediated processes. We suggest that proteins in the three categories above, formulated to trigger the appropriate mechanisms operating in rhesus macaques, would have both prophylactic and therapeutic potential as a human vaccine.     

Hormonal control of seasonal morphs by the timing of ecdysteroid release in Araschnia levana L. (Nymphalidae: Lepidoptera)

Araschnia levana L. occurs in two seasonal morphs. Larvae reared under short-day conditions become diapause pupae and emerge as red spring-morph butterflies. Long-day larvae become non-diapause pupae, which emerge as black and white summer morphs. Pupae reared under these different conditions were joined in parabiosis. Both underwent adult development without diapause and the long-day animals developed into the summer morph as normal. The morph of short-day animals depended on the time of parabiosis. When they were joined to fresh long-day pupae 1 day after their own pupation, summer morphs resulted. When parabiosis began 4 days after pupation or later, spring morphs resulted. Extirpation of the brain-corpora cardiaca-allata complex from long-day pupae affected neither non-diapause development nor the summer morph. Adult development could be prevented by removal of head and prothorax. When adult development was initiated in the remaining bodies by 20-hydroxyecdysone 14 days after pupation, the spring morph resulted.—Injection of 20-dydroxyecdysone into 3-day-old short-day pupae initiated adult development and led to the summer morph. Injections into 10-day-old short-day pupae led to the spring morph. The same was true in diapause pupae deprived of their brain-corpora cardiaca-allata complex. These results indicate that seasonal diphenism in A. levana is controlled only by the timing of ecdysteroid release, which initiates adult development. There is no direct influence of the brain on wing coloration.     

Characterization of the Arrest in Anther Development Associated with Gibberellin Deficiency of the gib-1 Mutant of Tomato

The role of gibberellins in flower bud development was investigated by studying the gib-1 mutant of tomato, Lycopersicon esculentum. This gibberellin-deficient mutant initiates flower buds, but floral development is not completed unless the mutant is treated with gibberellin. Treatment with other plant growth regulators does not induce normal flower development. Development of gib-1 flower buds, as measured by progress toward anthesis, ceases at a bud length of 2.5 millimeters; however, increase in size of the bud continues. Buds between 2.5 and 3.7 millimeters are developmentally arrested but still are capable of developing normally after treatment with gibberellic acid. Anthers of these developmentally arrested buds contain pollen mother cells that are in the G1 phase of premeiotic interphase. Following treatment of developmentally arrested buds with gibberellic acid, premeiotic DNA synthesis and callose accumulation in pollen mother cells are evident by 48 hours posttreatment, and within 66 hours, prophase I of meiosis- and meiosis-related changes in tapetum development are observable.     

Differential distribution of B16F10 melanoma cells in the liver lobule

The distribution pattern and the number of tumor cells arrested in the liver were studied in mouse livers. Mice were perfused intravascularly with a suspension of B16F10 melanoma cells. The animals were sacrificed at 0, 1, 5, and 20 min after tumor cell perfusion. The pattern of tumor cell distribution was studied by morphological methods, and by a combined method of fluorescent-tumor cell labelling and histochemical succinate dehydrogenase activity on frozen sections, in order to define the localization of tumor cells arrested in the liver lobule. The results show that the tumor cells have an exclusive distribution in the periportal regions of the liver lobule (identified as the high succinate dehydrogenase activity areas), and that the cells are not arrested in the pericentral regions (identified as the low succinate dehydrogenase activity areas). In addition, indomethacin treatment (2 mg/kg/day) induced an increase in the number of melanoma cells arrested in the liver, but a different distribution with respect to controls was not observed. These results show that periportal regions of the liver lobule constitute a particular domain in which the B16F10 melanoma cells present a special retention ability that can be modulated by indomethacin treatment.     

Investigation of the proteolipid protein promoter activity during demyelination and repair

The transgenic plp-GFP mouse line expressing the green fluorescent protein (GFP) driven by the mouse myelin proteolipid protein (plp) gene promoter has been previously used to study the contribution of the plp lineage to oligodendrocyte development in the embryonic brain. Here, we show that the GFP fluorescence reflects the developmental expression of proteolipid protein during the postnatal development until adulthood in brain slices and in primary cultures of plp-GFP⁺ cells derived from postnatal animals. In the adult brain, plp-GFP-expressing cells are mature oligodendrocytes but not oligodendroglial progenitors. In the model of focal demyelination induced by lysolecithin (LPC) in the corpus callosum of adult plp-GFP animals, we observed an up-regulation of the morphogen Sonic Hedgehog (Shh) in the LPC-induced lesion but not in the control animals. Moreover, we show that the adenovirus-mediated transfer of Shh in the lesion results in the attenuation of the demyelination extent as evidenced by GFP fluorescence analysis in Shh-treated and control animals. Altogether these data show how plp-GFP fluorescence can be monitored to follow the oligodendrocyte lineage during demyelination and identify Shh morphogen as an important factor during repair.     

Reproductive Growth and Dry Matter Production of Glycine max (L.) Merr. in Response to Oxygen Concentration

Reproductive as well as vegetative parameters of mature soybean (Glycine max [L.] Merr. cv. Wye) plants grown in chambers in which the aerial portion was exposed to altered pO₂ during all or part of the growth cycle were measured. Oxygen concentration was found to be a key factor controlling all phases of reproductive development. Exposure to 5% O₂ from early seedling stage to senescence increased leaf, stem, and root dry weights and reduced seed yields when compared to 21% O₂; exposure to low O₂ during the vegetative growth stage from early seedling to mid-flowering arrested pod but not seed development; exposure from mid-flowering to mid-pod filling almost completely arrested seed but not pod development; exposure from mid-pod filling to senescence arrested seed development at the mid-filling stage.