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Effects of calcium deficiency on chondrocyte hypertrophy and type X collagen expression in chick embryonic sternum.

Maintenance of chick embryos in long-term culture without their calcareous egg-shell is a useful method for studying the relationship between calcium homeostasis and cell differentiation during skeletogenesis. Previously, we have shown that in shell-less (SL) embryos, calcium deficiency induces a cartilage-like phenotype in osteogenic tissues, such as calvaria (Jacenko and Tuan [1986] Dev. Biol. 115:215). In this investigation, we have studied the relationship between cartilage calcification and hypertrophy, and the expression of type X collagen, a specific product of hypertrophic chondrocytes. For this study, the cephalic (calcifying) and caudal (permanently cartilaginous) regions of sterna from day 18 and day 20 normal (NL) and SL embryos were metabolically labeled with [14C]-proline. Analysis of the biosynthetic products revealed significant differences in type X collagen expression in the cephalic region of sternal cartilage. In NL tissues, type X collagen production increased from 13.1% of total collagen at day 18 to 43.7% at day 20. In contrast, in SL embryos, type X collagen was not detectable until day 20, when it represented only 1% of total collagen. Comparison of the NL and SL embryos with respect to their serum calcium level and sternal calcium content and histology revealed a direct relationship between low systemic calcium and limited cartilage hypertrophy, undermineralization, and decreased type X collagen production in the sternal cephalic cartilage. Supplementation of CaCO3 to SL embryos increased their serum and sternal calcium, and restored cartilage hypertrophy, mineralization, and type X collagen synthesis in the cephalic portion of the sterna. These findings confirm that a critical relationship exists between calcium homeostasis, chondrocyte hypertrophy, mineralization, and type X collagen synthesis in the cephalic region of sternal cartilage. These results further demonstrate the importance of calcium in the morphogenetic events of endochondral ossification, in particular the transition from hyaline cartilage to hypertrophic cartilage, and eventually to bone.

Animals↗

Morphological and ultrastructural aspects of the activation of avian medullary bone osteoclasts by parathyroid hormone.

The activation of physiologically inactive medullary bone osteoclasts by parathyroid hormone (PTH) was examined using light and electron microscopy and histomorphometric methods. Medullary bone osteoclasts are functionally inactive during the avian egg-laying cycle when an egg shell is not being calcified in the shell gland. Japanese quail hens were given 0.5 IU/g PTH and the medullary bone osteoclasts were examined up to 90 min later. Administration of PTH results in rapid changes in osteoclast morphology and ultrastructure. Within 10 min ectoplasmic regions containing condensed-appearing material are evident in areas of the cell adjacent to bone surfaces. In tannic acid-fixed specimens, these ectoplasmic regions contain bundles of filaments extending perpendicularly from the osteoclast plasma membrane into the cytoplasm. It is in these areas that ruffled border development is initiated. Even at 10 min after PTH administration, mineral crystals are seen between the developing cell surface invaginations and folds. By 15 min after PTH administration, ruffled borders have appeared next to bone surfaces. The rapid development of ruffled borders on medullary bone osteoclasts after PTH is confirmed by electron microscope histomorphometry. By 30 min after PTH administration, ruffled borders are well developed and large endocytic vacuoles are beginning to appear in the osteoclast cytoplasm. Light microscope histomorphometric measurements indicate that osteoclasts are also increasing in size and spreading along bone surfaces with time after PTH administration. This study provides a morphologic and ultrastructural description of osteoclast activation by PTH. The results indicate that osteoclasts may effect rapid changes in bone resorption and mineral metabolism due to exogenous PTH in hens.

Animals↗

A new technique for explantation and in vitro cultivation of chicken embryos.

A technique is described for explanting and cultivating chicken embryos in plastic drinking cups which have been modified with plastic wrap to reproduce the geometry and dimensions of the egg shell. Successful explantation rates of 97% are possible with a double-window technique, and survivability in cups exceeds that achievable in other in vitro systems (i.e., petri dishes). Long-term survival to the 21st day of incubation is seen routinely. This system with cups is less expensive than that with petri dishes, and simpler than that with plastic wrap/tripods. Thus, this new method of in vitro cultivation of chicken embryos improves upon explantation rate, survivability and system design, and has a wide range of applications in developmental biology, angiogenesis, cancer, and pharmacology research.

Animals↗

Chitin synthesis and degradation as targets for pesticide action.

Various pesticides are being used to destabilize, perturb, or inhibit crucial biochemical and physiological targets related to metabolism, growth, development, nervous communication, or behavior in pestiferous organisms. Chitin is an eukaryotic extracellular aminosugar biopolymer, massively produced by most fungal systems and by invertebrates, notably arthropods. Being an integral supportive component in fungal cell wall, insect cuticle, and nematode egg shell, chitin has been considered as a selective target for pesticide action. Throughout the elaborate processes of chitin formation and deposition, only the polymerization events associated with the cell membrane compartment are so far available for chemical interference. Currently, the actinomycetes-derived nucleoside peptide fungicides such as the polyoxins and the insecticidal benzoylaryl ureas have reached commercial pesticide status. The polyoxins and other structurally-related antibiotics like nikkomycins are strong competitive inhibitors of the polymerizing enzyme chitin synthase. The exact biochemical lesion inflicted by the benzoylaryl ureas is still elusive, but a post-polymerization event, such as translocation of chitin chains across the cell membrane, is suggested. Hydrolytic degradation of the chitin polymer is essential for hyphal growth, branching, and septum formation in fungal systems as well as for the normal molting of arthropods. Recently, insect chitinase activity was strongly and specifically suppressed by allosamidin, an actimomycetes-derived metabolite. In part, the defense mechanism in plants against invasion of pathogens is associated with induced chitinases. Chitin, chitosan, and their oligomers are able to act as elicitors which induce enhanced levels of chitinases in various plants. Lectins which bind to N-acetyl-D-glucosamine strongly interfere with fungal and insect chitin synthases. Plant lectins with similar properties may be involved in plant-pathogen interaction inter alia by suppressing fungal invasion.

Animals↗

Extraction and partial characterization of non-histone nuclear proteins of Schistosoma mansoni.

A pool of nuclear proteins from adult worms of Schistosoma mansoni was analyzed for amino acid composition and found to be compatible with high mobility group (HMG) proteins. One of the schistosome HMG proteins was identified as HMG 2 by one-dimensional and two-dimensional PAGE. Stage-specific differences in the HMG-like protein composition were encountered when adult worms were compared to schistosomula, the larval form. Immobilization of the adult male and female nuclear proteins onto nitrocellulose, followed by hybridization against 32P-F-10, a schistosome sex specific gene encoding a major egg shell protein, revealed distinct banding patterns. On the other hand, a synthetic oligonucleotide, derived from the 3' untranslated end of the F-10 gene and possibly containing one regulatory element of the gene, bound mainly to male low MW proteins.

Aging↗

Effects of feeding high dosages of vitamin E to laying hens on thyroid hormone concentrations of hatching chicks.

Long-term experimental feeding of 20,000 ppm alpha-tocopheryl acetate to laying hens caused a significant (P < 0.05) decrease in hatching rates as compared to the control group, which was fed a diet containing 19 ppm alpha-tocopherol. When the thyroid hormones in the developing chicks were checked on incubation days 16, 19, 21, and 22, the following results were ascertained: During the latter part of incubation, increases in plasma concentrations of thyroxine and triiodothyronine were observed. No significant differences in hormone concentrations (P > 0.05) between the control and the treatment group were observed during incubation days 16, 19, and 22. However, on the day of hatching (day 21 of incubation) significantly lower (P < 0.05) triiodothyronine concentrations in chick embryos of piped eggs were found in the treatment group. Moreover, thyroxine concentrations in non-piped eggs and in hatched chicks were found to be significantly higher as compared to the control group. Given these results, one concludes that extremely high dosages of vitamin E may affect thyroid hormone concentrations of hatching chicks, and therefore, the chicks might be inhibited in pipping the egg shell. Hypothetically, the hepatic enzyme 5'-monodeiodinase is involved in the mechanism of inhibition.

Animals↗

Water loss and survival in embryos of the domestic chicken.

In this report we show that the amount of water lost during incubation is an important determinant of hatchability in embryos of the domestic chicken. However, when the loss of water is restricted to the first half versus the second half of the total incubation period, it is the loss of water during the first half that affects hatching success. We propose that regulating the permeability of the egg shell may be important in preventing excessive drying of the inner shell membrane during early development along with limiting the total amount of water lost from the embryo during incubation.

Animals↗

Giant spermatozoon coiled in small egg: fertilization mechanisms and their implications for evolutionary studies on Ostracoda (Crustacea).

Ostracods of the superfamily Cypridoidea have giant spermatozoa. However, little data exist on the sperm-egg interaction in this group: only two publications have so far given the most ambiguous indication that the entire sperm enters the egg on fertilization. These assumptions have not yet been tested with modern techniques, nor has their putative value for developmental and evolutionary investigations been realized. The present paper gives the first, clear, light- and scanning electron microscopical evidence of the entire giant ostracod spermatozoon being incorporated into the egg. Coiling of the sperm underneath the egg shell is shown in the early zygotes of the species Mytilocypris praenuncia and Pseudocandona marchica. Additionally, data on the morphology of female and male reproductive tracts are given for M. praenuncia. Hypotheses on the evolution of giant filiform sperm in the Animal Kingdom are reviewed, and their applicability to ostracods is discussed. The demonstrated ingression of the entire sperm implies the entry of the two giant paternal mitochondrial derivates into the zygote in Cypridoidea and potentially casts doubt upon the dogma of strict maternal inheritance of mitochondrial DNA. Evidence of paternal inheritance of mtDNA in several organisms has recently given rise to a controversial debate on this issue; the possible significance of this phenomenon for molecular studies on ostracod phylogeny and evolution is discussed.

Animals↗

Embryonic integument and "molts" in Manduca sexta (Insecta, Lepidoptera).

In Manduca sexta the germ band is formed 12 h post-oviposition (p.o.) (=10% development completed) and is located above the yolk at the egg surface. The cells show a polar organization. They are engaged in the uptake and degradation of yolk globules, pinched off from the yolk cells. This process can be observed in the integumental cells during the first growth phase of the embryo that lasts until "katatrepsis," an embryonic movement that takes place at 40% development completed. At 37% development completed, the ectoderm deposits a thin membrane at its apical surface, the first embryonic membrane, which detaches immediately before katatrepsis. The second period of embryonic growth--from katatrepsis to 84 h p.o. (70% development completed)--starts with the deposition of a second embryonic membrane that is somewhat thicker than the first one and shows a trilaminar, cuticulin-like structure. Whereas the apical cell surface is largely smooth during the deposition of the first embryonic membrane, it forms microvilli during deposition of the second one. At the same time, uptake of formed yolk material ceases and the epidermal cells now contain clusters of mitochondria below the apical surface. Rough endoplasmic reticulum (RER) increases in the perinuclear region. The second embryonic membrane detaches about 63 h p.o. At 69 h p.o., a new generation of microvilli forms and islands of a typical cuticulin layer indicate the onset of the deposition of the larval cuticle. The third growth phase is characterized by a steady increase in the embryo length, the deposition of the larval procuticle, and by cuticular tanning at about 100 h p.o. Beginning at that stage, electron-lucent vesicles aggregate below the epidermal surface and are apparently released below the larval cuticle. Manduca sexta is the first holometabolous insect in which the deposition of embryonic membranes and cuticles has been examined by electron microscopy. In correspondence with hemimetabolous insects, the embryo of M. sexta secretes three covers at approximately the same developmental stage. A marked difference: the second embryonic cover, which in Hemimetabola clearly exhibits a cuticular organization, has instead a membranous, cuticulin-like structure. We see the difference as the result of an evolutionary reductional process promoted by the redundancy of embryonic covers in the egg shell. Embryonic "molts" also occur in noninsect arthropods; their phylogenetical aspects are discussed.

Animals↗

Ultrastructure of Mehlis' gland in the lung fluke, Paragonimus ohirai (Trematoda: Troglotrematidae).

Mehlis' gland of a digenetic trematode, Paragonimus ohirai, is composed of two types of secretory cells, DB and CB. The less abundant type (DB) produces dense bodies, with the cytoplasm characterized by greatly distended cisternae of rough endoplasmic reticulum. The other type (CB) synthesizes clear, vesicular bodies. Its cytoplasm contains numerous mitochondria, rough endoplasmic reticulum with narrow cisternae, and abundant Golgi complexes. Processes of the two cell types converge on the ootype-proximal uterine wall, pass through the epithelium, and finally open into the lumen. These proximal processes contain longitudinally arranged microtubules whose luminal ends are anchored to the epithelium by ring-form septate desmosomes. According to the distribution of the two types of processes, three different zones (DB, mixed, and CB) can be recognized within the epithelia. As the CB processes enter the lumen predominantly beyond the uterine valve region, this cell may produce secretions required for egg shell maturation or hardening. The role of DB cells (which enter the lumen more commonly in the ootype near the oviduct) remains unknown.

Animals↗

Identification and characterization of estrogen and progesterone receptors from the oviduct of the American alligator (Alligator mississippiensis).

A protein extract prepared from the egg shell fiber-secreting region of the oviduct of Alligator mississippiensis was assayed for the presence of receptors for estrogen and progesterone. In the presence of [3H]-estradiol-17 beta, the extract contained an estrogen-binding activity which reached equilibrium at 25 degrees C in 1 h. Scatchard analysis demonstrated that a single estrogen-binding activity was present in the extract with a Kd of 0.5 nM for [3H]estradiol-17 beta. A steroid specificity competition assay showed the estrogen binding activity strongly recognized estradiol-17 beta and diethylstilbestrol (DES) and weakly interacted with estrone, estriol, estradiol-17 alpha, and dihydrotestosterone (DHT). The estrogen binding activity did not recognize testosterone, dexamethasone or progesterone. The extract exhibited a DNA-binding activity that recognized an estrogen response element in a gel mobility shift assay. We have also identified a high affinity binding activity in the extract that specifically recognized the synthetic progestin R5020 with a Kd of 0.9 nM. This binding activity recognized 17 alpha-estradiol, dexamethasone, testosterone, and estriol. This activity did not recognize DHT, DES, or estradiol-17 beta. These data suggest the presence of estrogen and progesterone receptors in the oviduct of the alligator.

Alligators and Crocodiles↗

Ascaris suum: protein phosphotyrosine phosphatases in oocytes and developing stages.

Protein tyrosine phosphatases were analyzed in oocytes of Ascaris suum. Phosphatases dephosphorylating modified acidic lysozyme were present in high-molecular-weight form (M(r) > 600,000) and as a 50- to 55-kDa protein in the soluble fraction. The low-molecular-weight form of the phosphatase cross-reacted with an antiserum raised against human T-cell protein tyrosine phosphatase and was not distinguishable from the 50- to 55-kDa protein tyrosine phosphatase previously described in the muscular layer of the adult worms (B. Schmid et al. 1996, Molecular and Biochemical Parasitology 77, 183-192). The low-molecular-weight form was also present on immunoblots of high-molecular-weight protein tyrosine phosphatase preparations after denaturing electrophoresis. The same or a similar form of the tyrosine phosphatase was also found in detergent extracts from the pelletal fraction. In addition, another tyrosine phosphatase of 180 kDa molecular mass that dephosphorylated myelin basic protein was also found in extracts from the soluble compartment as well as in detergent extracts from the pelletal fraction. It showed no cross-reactivity with antisera raised against soluble mammalian phosphatases and was resistant to inhibition by vanadate. While the activities of the myelin basic protein-dephosphorylating protein phosphatase remained fairly constant during early development of the oocytes, the activity of the enzyme dephosphorylating modified lysozyme in the pelletal fraction decreased to less than 10% of the initial activity between days 3 and 28 of incubation. Immunocytochemical studies of unfertilized and developing Ascaris eggs revealed association of protein tyrosine kinase and protein tyrosine phosphatase with the egg shell, in addition to their presence in the neighborhood of mitochondria. The amount of enzyme changed with the stage of development. In the larval stage (21 days) protein tyrosine kinase had increased in the chitin layer of the shell and in the nuclei while the relative amount of tyrosine phosphatase decreased in accordance with the biochemical data.

Animals↗

On the molecular mechanism of intestinal calcium transport.

The intestinal absorption of calcium is certainly a complex process, dependent on several factors of which vitamin D, via 1,25(OH)2D3, is the major controlling hormone. The efficiency of calcium absorption is a function of calcium status and calcium need. As the body's demand for calcium increases, the process commonly termed, adaptation, is activated in which the synthesis of 1,25(OH)2D3 from precursor is increased, resulting in the stimulation of the rate of calcium absorption. The increased demand for calcium might result from the ingestion of a diet deficient in calcium, from growth, pregnancy, lactation and egg shell formation in the laying hen. Accomapanying the change in calcium absorptive efficiency are molecular modifications of the transporting enterocytes, some mentioned herein and elsewhere (Wasserman & Chandler, 1985; Wasserman, 1980; Wasserman et al., 1984). Highly correlated with the rate of calcium absorption under a wide variety of conditions is the concentration of the vitamin D-induced calcium-binding protein, calbindin-D28K (avian type) and calbindin-D9K (mammalian intestinal type). The role of calbindin-D in this transport process is not precisely known but is considered to act at the present time as a cytosolic facilitator of Ca2+ diffusion from the brush border membrane to the basolateral membrane. In addition to the induction of calbindin-D synthesis, 1,25(OH)2D3 exerts other effects on the intestinal epithelium that can have consequences on the calcium absorptive process. Some of these effects are summarized in Figure 14. Vitamin D-dependent reactions might be either direct effects of 1,25(OH)2D3 or indirect effects due to elevated intracellular Ca2+ concentrations. These include changes in the fluidity of the brush border membrane, an increase in microvillar alkaline phosphatase-low affinity Ca-activated ATPase activity, an association of calmodulin with the 105 kD brush border cytoskeletal protein and, following calbindin D synthesis, the binding of calbindin D to a 60 kD brush border protein and to microtubules. The latter has been suggested to be related to the proposed transfer of Ca2+ by an endocytotic-exocytotic mechanism. In addition, a vitamin D-dependent intestinal membrane calcium-binding protein has been identified (Kowarski & Schachter, 1980). Playing into this multi-component system is a stimulation of cyclic nucleotide synthesis by 1,25(OH)2D3 which, through activation of cyclic nucleotide-dependent protein kinases, might modify membrane Ca2+ "channels" by phosphorylation reactions.4+ Intracellular organelles, i.e., the endoplasmic reticulum, mitochondria, the Golgi apparatus, are potent sequesters of Ca2+ and could contribute to the protection of the cell from excessively high Ca2+ concentrations by transiently storing absorbed Ca2+.

Animals↗

Patterns, trends, and toxicological significance of chlorinated hydrocarbon and mercury contaminants in bald eagle eggs from the Pacific coast of Canada, 1990-1994.

Bald eagle (Haliaeetus leucocephalus) eggs were collected during incubation, 1990-1992, from 16 nests near three bleached-kraft pulp mills, from six nests in the Fraser River estuary and from seven nests at a reference site on the Pacific coast of Canada. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were present in all eggs in a qualitatively similar pattern among sites. Mean concentrations of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) were significantly higher in eggs collected from near three kraft pulp mill sites in the Strait of Georgia (44, 45, 84 ng/kg) than from the reference area in Johnstone Strait (15 ng/kg). There were few differences among sites in mean organochlorine pesticide levels, indicating the diffuse distribution of those chemicals and the domination of atmospheric inputs. Mean concentrations of total polychlorinated biphenyls (PCBs) were highest in eggs from the Strait of Georgia (4.86 mg/kg) and the PCB congener pattern was significantly different between that area and both the lower Fraser valley and Johnstone Strait. Mean mercury concentrations, which were mainly methyl-mercury, were significantly higher in eggs collected from the lower Fraser Valley (0.258 mg/kg) and Johnstone Strait (0.294 mg/kg) compared to the Strait of Georgia (0.188 mg/kg). Individual and regional variation in concentrations of organochlorine pesticides, PCBs and mercury in eagle eggs were thought to be influenced mainly by dietary differences. Toxicologically, in 1990, mean TCDD-toxic equivalents (TEQs) in bald eagle eggs were about two-fold greater than a lowest-observed-effect level, suggested elsewhere for this species, of 210 ng/kg TEQs. In the Strait of Georgia, PCCDs and PCDFs made a greater contribution to TEQs than non-ortho and mono-ortho PCBs, whereas the reverse was true for eggs outside the strait. Mean eggshell thickness was less than the pre-1947 value at all sites, although there was no significant relationship between eggshell thickness and DDE concentrations. Levels of other organochlorine pesticides and mercury were below those considered to be toxic.

Analysis of Variance↗

Histochemical and electron microscopy investigations on medullary bone.

Folliculin administration to pigeons stimulates the development of medullary bone in marrow spaces of the femora and other long bones. It is a specialized osseus tissue not devoted to mechanical functions and which is rapidly reabsorbed before egg-shell formation. Medullary bone is formed and reabsorbed in the same way as other types of bone. Consequently, because of its very rapid rate of formation and resorption, it represents an ideal tissue for studying osteoblastic, osteoclastic and osteocytic activity, and the calcification process. Medullary bone is deeply stained by PAS, Alcian blue and colloidal iron and is metachromatic after toluidine blue staining. This shows that its interfibrially ground substance contains relatively high amounts of glycoproteins and acid proteoglycans. Calcification initially occurs in maxtrix vesicles (or calcifying globules) which are very numerous between the collagen fibrils of the osteoid tissue, and successively spreads into the surrounding interfibrillar matrix. Here, the crystals are closely related to thin, filament-like organic structures which seem to be components of ground substance proteoglycans. These findings confirm that in medullary bone, as in other types of calcifying tissue, the inorganic substances is initially laid down within calcifying globules and is successively closely related to organic, non-collagenous, filamentous organic structures (crystal ghosts) which probably represent a framework for calcium salt deposition.

Animals↗

The hard parts (trophi) of the rotifer mastax do contain chitin: evidence from studies on Brachionus plicatilis.

The jaws (trophi) of the rotifer Brachionus plicatilis are soluble in strong acids but are resistant to long treatments by strong alkali. They show the same buoyant density as chitin and also as the chitin-containing layers of rotifer egg-shells. The presence of chitin in these structures was confirmed using the following techniques: chitosan-tests, thin-layer chromatography of trophi-hydrolysates which revealed glucosamine, by dissolving trophi with chitinase and electron microscopic WGA/gold-labelling. The content of chitin in the trophi was estimated by two different methods to be approx. 64% (50-75%).

Animals↗

Schistosomiasis at Loum, Cameroun; Schistosoma haematobium, S. intercalatum and their natural hybrid.

A survey of 500 schoolchildren in Loum in 1968 revealed an overall infection rate of 54.2% with Schistosoma intercalatum and this was the only species of schistosome encountered. In 1972 a number of children were found to be passing schistosome eggs in their urine and these eggs ranged in shape and size from the forms characteristic for S. haematobium to those of S. intercalatum. Preliminary laboratory studies demonstrated that hybridisation between the two species was occurring. Subsequent field surveys showed that the snail hosts for the two parasites (B. rohlfsi for S. haematobium and B. forskali for S. intercalatum) were both present in the river Mbette and its tributaries in Loum and the distribution of the two snail species coincided closely with the distribution of the schistosomes in the human population. Detailed study of a small group of children passing hybrid eggs in their urine revealed that few of them were passing eggs in their faeces and that those eggs which were found in faeces were not viable. Analysis of schistosome egg-shape by plotting cumulative size-frequency data on probability paper demonstrated that the graph obtained from a natural hybrid series was different from that given by a known mixture of the two separate species. The hybrid series included a number of exceptionally large eggs resembling those of S. bovis but isolation of these eggs and subsequent laboratory passage of the parasites showed that they were part of the series and were not evidence of the presence of a third species. Hybridisation experiments in the laboratory showed that the cross S. haematobium male X S. intercalatum femal is fully viable but that the reverse mating is not successful, thus accounting for the failure of the faecal eggs recovered from children with hybrid infections. Histological results from laboratory passaged hybrids suggest that the Ziehl-positive staining reaction of the egg-shells of S. intercalatum may be a recessive character. The observations reported here indicate that S. haematobium has only recently become established in Loum and that it is, through introgressive hybridisation, replacing the indigenous S. intercalatum. A suggested explanation for the change in the parasite fauna is offered and this depends upon ecological changes resulting from forest clearance and agricultural development providing improved conditions for the spread of B. rohlfsi, the snail host for S. haematobium. It is suggested that, in contrast to recent reports on the spread of S. intercalatum, this species is in fact retreating and being replaced by S. haematobium in areas where forest clearance is taking place. In conclusion it is suggested that introgressive hybridisation of this kind may have been responsible for the evolution of certain characteristic local strains of African schistosomes.

Bulinus↗