Obligatory Intermediate Host Research Articles

Combining a transcriptomic approach and a targeted metabolomics approach for deciphering the molecular bases of compatibility phenotype in the snail Biomphalaria glabrata toward Schistosoma mansoni

Biomphalaria glabrata is a freshwater snail and the obligatory intermediate host of Schistosoma mansoni parasite, the etiologic agent of intestinal Schistosomiasis, in South America and Caribbean. Interestingly in such host-parasite interactions, compatibility varies between populations, strains or individuals. This observed compatibility polymorphism is based on a complex molecular-matching-phenotype, the molecular bases of which have been investigated in numerous studies, notably by comparing between different strains or geographical isolates or clonal selected snail lines. Herein we propose to decipher the constitutive molecular support of this interaction in selected non-clonal resistant and susceptible snail strain originating from the same natural population from Brazil and thus having the same genetic background. Thanks to a global RNAseq transcriptomic approach on whole snail, we identified a total of 328 differentially expressed genes between resistant and susceptible phenotypes among which 129 were up-regulated and 199 down-regulated. Metabolomic studies were used to corroborate the RNAseq results. The activation of immune genes and specific metabolic pathways in resistant snails might provide them with the capacity to better respond to parasite infection.

Hemocytes from neonates of Biomphalaria glabrata are functionally less mature than those from adult snails

AbstractGastropod molluscs, which serve as obligatory intermediate hosts for digenetic trematodes, possess an internal defense system (IDS) consisting of phagocytic hemocytes and plasma factors. This IDS is responsible for resistance to infection with larval trematodes, which are encapsulated and killed by hemocytes in incompatible snails. Like other physiological systems, the IDS probably undergoes maturation during early stages of life, and the relatively undeveloped state of the IDS in young snails has been hypothesized to be a factor in their increased susceptibility to infection with larval trematodes. In this study, hemocytes were examined in the BS‐90 laboratory strain of Biomphalaria glabrata that is resistant to infection with Schistosoma mansoni as adults but susceptible to infection as neonates. Compared with hemocytes from adults, hemocytes from neonates had a smaller perimeter and lower intrinsic directional motility on glass microscope slides. Additionally, in vitro assays showed a lower association with fucoidan‐linked polystyrene beads and less ability to produce superoxide anion in hemocytes from neonates compared to hemocytes from adults. These results support the hypothesis that the gastropod IDS undergoes maturation during growth. However, whether the observed differences between hemocytes of neonatal and adult BS‐90 snails play a role in the susceptibility of the former and resistance of the latter to infection with S. mansoni is not known.

Morphology and activities of cell populations of haemocytes in Oncomelania hupensis following Schistosoma japonicum infection

Oncomelania hupensis is the only obligatory intermediate host of Schistosoma japonicum, the pathogen of zoonosis schistosomiasis. Haemocytes play a critical role in the cellular immune defence of O. hupensis against S. japonicum challenge. Here, the morphology and classification of haemocytes of O. hupensis were investigated by Giemsa staining and light microscopy, combining with the scanning and transmission electron microscopy and flow cytometry. Granulocytes and hyalinocytes were confirmed as two main types of haemocytes, account for ~ 10% and ~ 90% of all haemocytes, with size varying in 4.3–10.9 μm and 0.4–30.8 μm, respectively. Subpopulations can be identified further by granule feature, shape, size, and surface and inner structure of cells. The heterogeneity in morphology implied varied developmental process and function of haemocyte subpopulations. After the S. japonicum challenge, haemocytes of O. hupensis respond to S. japonicum invasion immediately. The dynamic change of haemocyte subpopulations indicates that the small hyalinocyte could differentiate into a larger one or granulocyte after S. japonicum challenge, and the granulocytes and larger hyalinocytes play leading roles in early defence reaction, but in different ways. Phagocytosis and apoptosis of haemocytes in O. hupensis were proved to be related to immune defence against S. japonicum, with the combined effect of granulocytes and larger hyalinocytes. However, the main pathway of each subpopulation to take effect in different periods need further investigation.

Distribution of Biomphalaria Snails in Associated Vegetations and Schistosome Infection Prevalence Along the Shores of Lake Victoria in Mbita, Kenya: A Cross-Sectional Study

Background: Schistosomiasis due to Schistosoma mansoni remains a major public health problem and cause of morbidity and mortality in sub-Saharan Africa despite the implementation of control programmes. More than 6 million Kenyans are at risk of infection. Regarding control measures, Biomphalaria snail species, which are the obligatory intermediate hosts for transmission of S. mansoni, have been neglected. Mbita subcounty in Homa Bay County, western Kenya, along Lake Victoria basin, has a high prevalence of S. mansoni infection despite mass drug administration. This study aimed to determine the abundance of Biomphalaria, with their associated vegetation and schistosome infection rates, along Mbita shoreline. Methods: Sixteen purposively selected sites along the Mbita shoreline were sampled for Biomphalaria snails using a 30-minute scooping technique. Global positioning system technology was used to map selected sites. The associated vegetation at sampling sites were collected and identified. Schistosome infection status among the snails was determined via the detection of cercaria shedding. Results: A total of 3,135 Biomphalaria sudanica snails were collected. The number of snails collected differed significantly between the 16 sites (F=11.735; degrees of freedom [df]=15.836; P<.001). Significant mean differences (MD) were also observed in terms of the number of snails collected per vegetation type (F=7.899; df=5.846; P<.001). The mean number of snails collected from Cyprus gracilis was significantly higher than that from Enydra fluactuants (MD= 2.03; P<.001), Eichhornia crassipes (MD=4.15; P<.010), and E. fluactuants mixed with E. crassipes (MD=2.516; P<.010). A total of 21 (0.67%) snails shed human cercariae, while 27 (0.86%) snails shed nonhuman cercariae, despite 14 sites having human faeces contamination. Conclusion: Although the schistosome infection prevalence among the snails was low, these sites may still be important exposure sites. C. gracilis is the main vegetation type associated with a high abundance of Biomphalaria snails. Molecular techniques are necessary for verification of schistosome positivity among the snails.

Space and time predictions of schistosomiasis snail host population dynamics across hydrologic regimes in Burkina Faso.

The ecology of the aquatic snails that serve as obligatory intermediate hosts of human schistosomiasis is driven by climatic and hydrological factors which result in specific spatial patterns of occurrence and abundance. These patterns in turn affect, jointly with other determinants, the geography of the disease and the timing of transmission windows, with direct implications for the success of control and elimination programmes in the endemic countries. We address the spatial distribution of the intermediate hosts and their seasonal population dynamics within a predictive ecohydrological framework developed at the national scale for Burkina Faso, West Africa. The approach blends river network-wide information on hydrological ephemerality which conditions snail habitat suitability together with ensembles of discrete time ecological models forced by remotely sensed estimates of temperature and precipitation. The models were validated against up to four years of monthly snail abundance data. Simulations of model ensembles accounting for the uncertainty in remotely sensed products adequately reproduce observed snail demographic fluctuations observed in the field across habitat types, and produce national scale predictions by accounting for spatial patterns of hydrological conditions in the country. Geospatial estimates of seasonal snail abundance underpin large-scale, spatially explicit predictions of schistosomiasis incidence. This work can therefore contribute to the development of disease control and elimination programmes.

Spirocercosis in Dogs in Israel

Abstract Spirocerca lupi is a nematode causing spirocercosis disease that affects mostly carnivores and especially canidae. The life cycle of S. lupi includes a coprophagous beetle as an obligatory intermediate host and a variety of facultative paratenic hosts. In Israel, spirocercosis is considered to be a serious condition with a variety of clinical signs comprising a great risk to canine populations. The diagnosis at an early infective stage is unreliable and the vast majority of infected dogs are diagnosed only when the disease has advanced. In advanced stages of the disease, treatment is difficult and there is a high risk for complications. A study was carried out to compare the prevalence of S. lupi in the central region of Israel with a previous investigation and by that consequently try to estimate the efficacy of preventative treatment used nowadays in Israel. The study was done by the use of two different methods: looking for the infective larvae (L3) in the main intermediate host in Israel, Ontophagus sellatus, and searching for the eggs of S. lupi by performing flotation methods on faecal samples. Beetles and faecal samples were collected from four different locations in the winter and summer of 2017, 2018, and 2019. According to the literature review and collection of data from case studies, the prevalence of spirocercosis is increasing in Israel, despite the negative results from the dissections of beetles and faecal samples.

Distribution of Biomphalaria Snails in Associated Vegetations and Schistosome Infection Prevalence Along the Shores of Lake Victoria in Mbita, Kenya: A Cross-Sectional Study.

Background:Schistosomiasis due to Schistosoma mansoni remains a major public health problem and cause of morbidity and mortality in sub-Saharan Africa despite the implementation of control programmes. More than 6 million Kenyans are at risk of infection. Regarding control measures, Biomphalaria snail species, which are the obligatory intermediate hosts for transmission of S. mansoni, have been neglected. Mbita subcounty in Homa Bay County, western Kenya, along Lake Victoria basin, has a high prevalence of S. mansoni infection despite mass drug administration. This study aimed to determine the abundance of Biomphalaria, with their associated vegetation and schistosome infection rates, along Mbita shoreline.Methods:Sixteen purposively selected sites along the Mbita shoreline were sampled for Biomphalaria snails using a 30-minute scooping technique. Global positioning system technology was used to map selected sites. The associated vegetation at sampling sites were collected and identified. Schistosome infection status among the snails was determined via the detection of cercaria shedding.Results:A total of 3,135 Biomphalaria sudanica snails were collected. The number of snails collected differed significantly between the 16 sites (F=11.735; degrees of freedom [df]=15.836; P<.001). Significant mean differences (MD) were also observed in terms of the number of snails collected per vegetation type (F=7.899; df=5.846; P<.001). The mean number of snails collected from Cyprus gracilis was significantly higher than that from Enydra fluactuants (MD= 2.03; P<.001), Eichhornia crassipes (MD=4.15; P<.010), and E. fluactuants mixed with E. crassipes (MD=2.516; P<.010). A total of 21 (0.67%) snails shed human cercariae, while 27 (0.86%) snails shed nonhuman cercariae, despite 14 sites having human faeces contamination.Conclusion:Although the schistosome infection prevalence among the snails was low, these sites may still be important exposure sites. C. gracilis is the main vegetation type associated with a high abundance of Biomphalaria snails. Molecular techniques are necessary for verification of schistosome positivity among the snails.

Regional report on Angiostrongylus vasorum in Colombia: Genetic similarity to European lineage.

The canine lungworm Angiostrongylus vasorum is considered neglected in South America and was only sporadically reported in dogs and wildlife. Gastropods act as obligatory intermediate hosts for this parasitosis. We here analysed Achatina fulica (African giant snail) populations from 5 regions of Colombia for A. vasorum infections. In total, 609 snails were collected from the departments Antioquia, Valle del Cauca and Putumayo. Angiostrongylus vasorum-infected A. fulica were found in all departments with a total prevalence of 3.9%. Larvae originating from Putumayo were molecularly characterized and identified as the European lineage of A. vasorum. This regional report shows for the first time the presence of A. vasorum in intermediate hosts in Colombia and the European genotype in South America.

The lifecycle of Dichelyne minutus (Rudolphi, 1819) (Nematoda: Cucullanidae) in the estuarine biocenosis of the Black Sea

The lifecycle, the host–parasite system, and the ecological features of the nematode Dichelyne minutus (Rudolphi, 1819), which parasitizes invertebrates and fish in the estuarine biocenosis located at the influx of the Chornaya River into the Black Sea (off Sevastopol), have been studied. The host–parasite system of D. minutus includes the polychaete Hediste diversicolor Muller, 1776 (as an obligatory intermediate host) and nine fish species, of which seven are definitive hosts and two are accidental or captive hosts. It has been found that the lifecycle of D. minutus in the biocoenosis of the Black Sea differs from the lifecycle of this nematode that inhabits the Baltic and North seas. In the studied biocoenosis, nematode larvae occur in polychaetes and fish only in the spring and summer; no larvae are found in the autumn (the study was not conducted in the winter). The nematode parasitizes the polychaete H. diversicolor in the spring; the main source of infection in this period is obviously nematode eggs that were laid in the autumn and have overwintered in the environment. The infection process ends by early summer. The seasonal and size–age dynamics of nematode infection of the round goby, Neogobius melanostomus (Pallas, 1814), are analyzed taking the specifics of fish biology into account. The short period of infection, as characterized by the active emission of nematode larvae, their low survival in polychaetes and fish, a short lifecycle and the mortality of mature nematodes after egg-laying in the autumn result in an over-scattered distribution (mostly of the negative-binomial type) of D. minutus in populations of all the hosts.

A pilot study to determine the prevalence of human rat lungworm (Angiostrongylus cantonensis) exposure on Hawaii Island

Abstract The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen that is the cause of human eosinophilic meningitis and ocular angiostrongyliasis. It is considered as a global, emerging infectious disease commonly known as rat lungworm disease (RLWD). Rats are the definitive hosts, mollusks are the obligatory intermediate hosts. Humans can become infected by ingesting intermediate hosts containing infective larvae in contaminated food or water. There are currently no diagnostic tests for RLWD available in Hawaii and prevalence of infection is unknown. To estimate prevalence of exposure to RLW parasites, working with Clinical Labs of Hawaii, LLP and Puna Community Medical Center in Pahoa, HI, we collected serum from 436 human volunteers from east Hawaii where most of the cases of RLWD in the USA originate. The presence of RLW antibodies were evaluated by indirect ELISA using crude antigen extracted from adult A. cantonensis nematodes. Of the 360 samples successfully analyzed, 76 (21%) were strongly positive, 166 (46%) weakly positive and 118 (33%) negative. Based on self-reported data, 92 (26%) believed they have or had RLWD, and 15 (4.2%) were definitively diagnosed. 77% of volunteers reported catchment systems as their main source of household water. More studies would need to be completed to evaluate the potential for water transmission of RLWD. Due to the potential for false positives using crude RLW antigen, results will be verified using dot-blots containing a 31 kDa protein which is expected to provide greater specificity.

Analysis of Circulating Haemocytes fromBiomphalaria glabratafollowingAngiostrongylus vasorumInfection Using Flow Cytometry

Angiostrongylus vasorum is an emerging parasite of dogs and related to carnivores that have an indirect life cycle, with a wide range of terrestrial and aquatic gastropods as the obligatory intermediate host. Unfortunately, the relationship between A. vasorum and their snail hosts remains poorly understood. Circulating haemocytes are the main line of cellular defence involved in the destruction of helminths in snails. Aiming to further characterize the haemocyte subsets in Biomphalaria snails, we have performed a flow cytometric analysis of whole haemolymph cellular components using a multiparametric dual colour labelling procedure. Our findings demonstrated that B. glabrata infected with A. vasorum have two major circulating haemocyte subsets, referred to as small and large haemocytes. Differences in the cell proportion occurred over time. The development of better invertebrate infection control strategies would certainly result in the better control of human diseases caused by other species of the genus Angiostrongylus. Such knowledge will assist in the establishment of novel control strategies aimed at parasites that use molluscs as intermediate hosts and clarify new aspects of the parasite-host relationship regarding cell recognition and activation mechanisms, which are also found in the innate response of vertebrates.

Re-establishment of the fish parasite fauna in the Tisa River system (Slovakia) after a catastrophic pollution event

An extensive survey of helminth parasites in 1,316 freshwater fish representing 31 species from two aquatic ecosystems in southeastern Slovakia characterized by different level of environmental pollution was carried out. The helminth species diversity was compared between the Tisa River, heavily polluted with cyanides and heavy metals after a series of ecological disasters in 2000, and the less anthropogenically influenced Latorica River. A parasitological survey found 31 gastrointestinal helminths: Trematoda (11 species), Cestoda (14), Acanthocephala (3) and Nematoda (3). As many as 70 host-parasite combinations have been found. Twenty of them (28.6%) represent new host-parasite finding records for the territory of Slovakia. The component communities were species-poor in both rivers, with high dominance of one to three helminth species. Even though the fish communities were qualitatively similar (ICS = 81%) and the number of fish examined was approximately the same (676 and 640) in both localities, the helminth species richness and diversity of host-parasite combinations were two times lower in the more polluted Tisa River. The helminth communities were also much less abundant in the Tisa River. Based on the Czekanowski-Sørensen similarity index (ICS = 48.8%) and the Percentage similarity index (PI = 19.5%), the helminth communities were qualitatively and quantitatively different in the two rivers. The remarkable lack of species diversity in the Tisa River can be explained by the negative impact of residual contamination of the Tisa river bottom on certain freshwater invertebrates (bivalves and prosobranch mollusks, copepods and amphipods) which serve as obligatory intermediate hosts for the helminths. Four species, the aspidogastrean Aspidogaster limacoides Diesing, 1835, the acanthocephalan Pomphorhynchus tereticollis (Rudolphi, 1809) tapeworms Atractolytocestus huronensis Anthony, 1958 and Khawia sinensis Hsü, 1935 are recorded in Slovakia for the first time.

Comparative pathology of parasitic infections in free-ranging and captive pit vipers (Bothrops jararac)

Between June 1997 and May 1998,47 pit vipers (Bothrops jararaca) (Group A) were euthanased when they were brought to the Instituto Butantan by farmers, and examined postmortem; during the same...

Hymenolepis microstoma: direct life cycle in immunodeficient mice.

The mouse bile duct tapeworm Hymenolepis microstoma requires beetles as the obligatory intermediate host. However, when congenitally athymic NMRI-nu mice were infected with the mature tapeworm and allowed to eat their own faeces with tapeworm eggs, the oncospheres penetrated the intestinal tissue and developed to cysticercoids. After excysting, growth to adult worms occurs in the lumen of the small intestine and bile duct. Furthermore, the same happened when NMRI-nu mice, non-obese diabetic severe combined immunodeficiency (NOD/Shi-scid) mice and NOD/Shi-scid, IL-2 Rgamma(null) (NOG) mice were orally inoculated with shell-free eggs of this parasite. Differences between the cysticercoids of H. microstoma and H. nana developed in the mouse intestinal tissues were: (i) the time course for the development of fully matured cysticercoids of H. microstoma in mice was about 11 days but only 4 days for H. nana; and (ii) cysticercoids of H. microstoma developed in mice had a tail while those of H. nana had none.

The life cycle of Dichelyne (Cucullanellus) minutus (Nematoda: Cucullanidae)

Mature specimens of the nematode Dichelyne (Cucullanellus) minutus (Rudolphi, 1819) (Ascaridida, Cucullanidae, Seuratoidea) were obtained from the intestine of flounder Platichthys flesus (L.) caught in the Oresund, Denmark. Plaice Pleuronectes platessa L. and common goby Pomatoschistus microps (Kröyer) also harbour this species. The eggs embryonate on the seabottom. Larvae about 440 microm long, and believed to be in their third stage, hatch from the eggs. These larvae are not directly infective to flounders or gobies. The polychaete Nereis diversicolor O.F. Muller acts as obligatory intermediate host. Experimental infections showed that larvae >600 microm long and provided with achitinous tooth survived in flounder and common goby. The third-stage larvae moult to fourth-stage larvae in the fish gut wall. Mature worms occur in the lumen of the anterior part of the intestine. All developmental stages may be transferred from one flounder to another; thus the flounder may acquire the parasite also by devouring infected gobies.

Complexity in Parasite Life Cycles: Population Biology of Cestodes in Fish

1. This paper examines the population biology of two related species of bothriocephalid cestodes, parasites of teleostean fish, which live in sympatry. One species, Bothriocephalus barbatus, needs in its life cycle only one obligatory intermediate host, a copepod. The second species, B. gregarius, needs the same intermediate host first, but also utilizes a non-obligatory paratenic host, which is a gobiid fish. In the case of B. gregaring, definitive hosts can be infested via the intermediate host or the paratenic host. A simple mathematical model was built to investigate the effect of the addition of a paratenic host into the life cycle of a parasite. 2. Results of the simulation clearly demonstrated that the maintenance or absence of infectivity of cestode larvae in the paratenic host could explain the observed levels of infection in both definitive hosts. 3. Acquisition of paratenic hosts has two advantages: the recovery of lost infective stages in a previously non-suitable intermediate host, and an increase in the time of infection during which the definitive host could be infected as the result of eating copepods in its planctonophagous juvenile existence and by eating gobies in its predaceous older stages. 4. Using the basic transmission rate as a measure of fitness, we also investigated the possibility of maturation of B. gregarius in the paratenic host, e.g. the acquisition of a new definitive host by the parasite. Basic transmission rates and numerical simulations suggest that there is no benefit for the parasite in evolving towards this strategy

Experimental life cycle of Lagochilascaris minor Leiper, 1909.

The life cycle of Lagochilascaris minor was studied using material collected from human lesion and applying the experimental model: rodents (mice, hamsters), and carnivores (cats, dogs). In mice given infective eggs, orally, hatch of the third stage larvae was noted in the gut wall, with migration to liver, lungs, skeletal musculature and subcutaneous tissue becoming, soon after, encysted. In cats infected with skinned carcasses of mice (60 to 235 days of infection) it was observed: hatch of third stage larvae from the nodules (cysts) in the stomach, migration through the oesophagus, pharynx, trachea, related tissues (rhino-oropharynx), and cervical lymph nodes developing to the mature stage in any of these sites on days 9-20 post inoculation (P.I.). There was no parasite development up to the mature stage in cats inoculated orally with infective eggs, which indicates that the life cycle of this parasite includes an obligatory intermediate host. In one of the cats (fed carcass of infected mice) necropsied on day 43 P.I., it was observed the occurrence of the self-infective cycle of L. minor in the lung tissues and in the cervical region which was characterized by the finding of eggs in different stages of development, third stage larvae and mature worms. It's believed that some component of the carnivore gastrointestinal tracts may preclude the development of third stage larvae from L. minor eggs what explains the interruption of the life cycle in animals fed infective eggs. It's also pointed out the role of the intermediate host in the first stages of the life cycle of this helminth.

Helminths of the arctic fox, Alopex lagopus (L.), in Greenland

Eight species of intestinal helminths were obtained from 38 arctic foxes, Alopex lagopus (L.), from two localities in Greenland (Thule Air Base and Søndre Strømfjord), viz., Echinoparyphium sp., 13%; Plagiorchis elegans (Rudolphi, 1802), 45%; Cryptocotyle concavum (Creplin, 1825), &lt;3%; Diphyllobothrium dendriticum (Nitzsch, 1824), 10%; Taenia ovis krabbei Moniez, 1879, 13%; Mesocestoides lineatus (Goeze, 1782), 13%; Toxascaris leonina (von Linstow, 1902), 39%; and Uncinaria stenocephala (Railliet, 1884), &lt;3%. New host and geographic records are reported. Cestodes for which rodents are obligatory intermediate hosts were not recorded, suggesting that the series of foxes was drawn from local populations inhabiting the western coast of Greenland. Helminth–host interactions are briefly discussed.

TRANSMISSION OF PIG LUNGWORMS (METASTRONGYLUS SP.).

THE life-cycle of the pig lungworm (Metastrongylus sp.) has been known since Hobmaier and Hobmaier1 incriminated earthworms as the obligatory intermediate host. Lungworm infestation in pigs is therefore likely to be a problem only where animals have access to pasture and hence earthworms.

Studies on the Taxonomy and Life History of Echinocephalid Worms (Nematoda : Spiruroidea) with a Complete Description of Echinocephalus pseudouncinatus Millemann, 1951

Third-stage, molting third-stage, and fourth-stage juveniles, and young and mature adults of Echinocephalus pseudouncinatus Millemann, 1951 from the homed shark, Heterodontus francisci, and the bat stingray, Myliobatis californicus, from the Gulf of California, Mexico, are described. During the third molt the number of rows of hooks on the head increases from 8 to the definitive number of approximately 19. The taxonomy of the genus is discussed, and E. spinosissimus (von Linstow, 1905) is reduced to synonymy with E. uncinatus Molin, 1858. The type species E. uncinatus is redescribed and the generic definition is emended. A key to the species of Echinocephalus is presented. The life history of echinocephalids is discussed. It is concluded that the definitive hosts, elasmobranch fishes, may become infected either directly by the ingestion of encysted juveniles in the obligatory intermediate hosts, various mollusks, or indirectly by the ingestion of similar juveniles in transport hosts, various teleost fishes. Millemann (1951) described a new species of Echinocephalus, E. pseudouncinatus, based solely on juvenile worms encysted in the foot of the pink abalone, Haliotis corrugata, from San Clemente Island, California. In 1953 Dr. Clark P. Read collected a large number of echinocephalids from elasmobranchs in the Gulf of California, Mexico, and these specimens were kindly made available to the writer for study. Examination of this material has resulted in the following findings: (1) validation of the species E. pseudouncinatus and extension of the species description to include adult and juvenile stages; (2) clarification of the taxonomy of the genus; and (3) clarification of the life history of these parasites. The present paper is concerned with these observations.