Schistosoma mansoni: relationship between cercarial production levels and snail host susceptibility

Two populations of Biomphalaria glabrata snails differing slightly in their susceptibility to Schistosoma mansoni infection showed dramatic differences in cercarial output per snail. Exposed to five or more miracidia, snails from a group with a 90-100% susceptibility rate (Group A) produced nearly twice the number of cercariae as those from a group with a 70-80% susceptibility rate (Group B). Exposure of individual snails to known numbers of miracidia resulted in higher numbers of primary (mother) sporocysts in Group A snails than in Group B snails. However, monomiracidial exposure of snails from both groups resulted in equivalent numbers of cercariae produced per positive snail, indicating that, once established, all primary sporocysts possess a similar reproductive potential. Morphometric analysis of serially sectioned 9-day-old primary sporocysts supported this conclusion; the size of the primary sporocysts and the size and numbers of secondary (daughter) sporocysts within each primary sporocyst were comparable in snails from both groups. The data indicate cercarial production in this system is regulated prior to, and/or during, early development of the primary sporocyst.     

Germinal elements and their development in Echinostoma caproni and Echinostoma paraensei (Trematoda) miracidia.

Among the large cells located in the posterior of Echinostoma caproni and E. paraensei miracidia are secretory cells, germinal cells (GC), and undifferentiated cells. Secretory cells do not give rise to progeny, whereas GC do. Undifferentiated cells develop into GC that can also divide to produce embryos. Cleavage of GC of E. caproni occurs only after the parasite has entered the snail host and develops into a sporocyst. With E. paraensei, GC are larger than noted for E. caproni, and in 3 of 23 miracidia examined, germinal cell cleavage had occurred in the miracidium such that an embryo containing 20-25 blastomeres was present. Observations on the germinal elements of miracidia help to explain previous results showing that (1) E. paraensei sporocysts release mother rediae a few days earlier than do sporocysts of E. caproni, and that (2) a single mother redia is produced ahead of all others by sporocysts of E. paraensei, but not by sporocysts of E. caproni. The present study adds support to the concept that E. caproni and E. paraensei are distinct species.     

Schistosoma mansoni: origin and expression of a tegumental surface antigen on the miracidium and primary sporocyst

A monoclonal antibody, recognizing a carbohydrate epitope associated with several tegumental surface components on Schistosoma mansoni primary sporocysts, was used to follow tegumental formation during transformation of the miracidium to sporocyst and its subsequent development in vitro and in vivo. Indirect fluorescent antibody and direct immunogold labeling methods confirm a structural connection between the intercellular ridges and a submuscular, multinucleate syncytium in the miracidium. Immunoreactive vesicles within this latter system directly contribute to elaboration of the tegumental surface membrane, through the process of membrane fusion. Lateral expansion of intercellular ridges by vesicular fusion ultimately result in fully transformed sporocysts exhibiting vesicular membrane epitopes as prominent tegumental surface components. Light microscopical and ultrastructural observations, together with Western immunoblot analyses, suggest a gradual depletion of intracellular and surface immunoreactive material of vesicular origin in primary sporocysts grown in culture for up to 12 days. In contrast, similar immunoreactive vesicles appear to be continuously synthesized throughout in vivo primary sporocyst development. Monoclonal antibody reactive epitopes appear to be uniquely expressed in the miracidium/primary sporocyst since similar molecules are absent from daughter sporocysts, cercariae, adults, and snail tissues.     

日本血吸虫毛蚴对钉螺的钻穿及在螺体内的分布和移行

采自安徽省池的钉螺每粒感染５０只湖南株日本血吸虫毛后的组织学观察说明：毛蚴钻穿钉螺有从螺鳃部、头足总后有皮以及实质组织（外套膜、触角和阴茎）等三方面途径,其中以前二者尤为重要;毛蚴进入螺鳃丝后直接进入血液循环系统,从头足表皮进入的毛蚴,除了少数在钻穿部位附近滞留外,多数继续向头足部深层的肌肉和窦状组织间隙移行,以前头足窦、直肠和消化道外的组织间隙以及肾脏为主要的移行部位;从外套膜、触角、阴茎等部位     

The invasion of Biomphalaria pfeifferi by Schistosoma mansoni miracidia and the development of daughter sporocysts

Laboratory experiments have been carried out to determine the susceptibility of Gezira Biomphalaria pfeifferi snails to S. mansoni miracidia and the relationship between miracidia and daughter sporocyst production at the 10–17 day development stage. The relationship between snail numbers, miracidia numbers and water volume has also been studied. Two non susceptible snails, Bulinus truncatus and Cleopatra bulimoides, both of which occur naturally in Gezira canals, were tested to see if they act as decoys for S. mansoni miracidia.The results showed that the B. pfeifferi are 100% susceptible to S. mansoni invasion, at least to the daughter sporocyst development stage. The more miracidia that penetrated the more daughter sporocysts were produced, however individual variation and overlap were great. When one miracidium was released to find one snail it succeeded in low water volumes (5 m, 50 ml), but failed in 5 litres. When 100 miracidia were released mortality of snails was high suggesting superinfection particularly when only one or five snails were available. Among survivors daughter sporocyst counts were very high. Cleopatra and Bulinus snails do have a decoy effect when present in large numbers. In their presence the number of infected snails was marginally reduced and the number of daughter sporocysts greatly reduced. However, if superinfection is reduced by decoy effect, it is conceivable that Biomphalaria may be protected by decoy snails in circumstances where miracidia counts are high.     

Lectin-binding properties of the surfaces of in vitro-transformed Schistosoma mansoni and Echinostoma paraensei sporocysts

As carbohydrates on the surfaces of sporocysts of digenetic trematodes may be targets of attack by the molluscan internal defense system, the lectin-binding patterns of living, in vitro-transformed sporocysts of Schistosoma mansoni and Echinostoma paraensei were characterized. Schistosoma mansoni sporocysts specifically bound 8 and E. paraensei 6 of 11 lectins examined. Sporocysts of the 2 species responded differently to 7 of the 11 lectins. Lectins inhibitable by mannose, galactose, and N-acetylgalactosamine were bound by both species. Lectins inhibited by fucose and N-acetylglucosamine bound uniquely to S. mansoni, and an N-acetylneuraminic acid (NeuNAc)-inhibitable lectin bound only to E. paraensei. Preincubation of sporocysts of either species in the plasma of the host snail Biomphalaria glabrata for as long as 24 hr only marginally altered the subsequent binding of lectins. Pretreatment of S. mansoni sporocysts with pronase E and trypsin substantially altered subsequent lectin binding, but similar treatment of E. paraensei sporocysts had little effect. A neuraminidase enzyme derived from Clostridium perfringens diminished binding of the NeuNAc-inhibitable lectin to E. paraensei sporocysts. This study indicates that lectin-binding monosaccharides are expressed abundantly on sporocyst surfaces, they vary considerably between 2 species parasitizing the same host, and they are not obscured readily or altered by exposure to host plasma.     

Localization of carbohydrate determinants common to Biomphalaria glabrata as well as to sporocysts and miracidia of Schistosoma mansoni

The presence of antigenic carbohydrate epitopes shared by Biomphalaria glabrata as well as by the sporocysts and miracidia representing snail-pathogenic larval stages of Schistosoma mansoni was assayed by immunohistochemical staining of paraformaldehyde-fixed tissues. To this end, both polyclonal rabbit antiserum raised against soluble egg antigens (SEA) of S. mansoni and monoclonal antibodies recognizing the carbohydrate epitopes LDN [GalNAc(beta1-4)GlcNAc(beta1-)], F-LDN [Fuc(alpha1-3)GalNAc(beta1-4)GlcNAc(beta1-)], LDN-F [GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-)], LDN-DF [GalNAc(beta1-4)[Fuc(alpha1-2)Fuc(alpha1-3)]GlcNAc(beta1-)] and Lewis X [Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-)] were used. Intriguingly, anti-SEA serum as well as anti-F-LDN antibodies displayed significant binding in the foot region, anterior tissue and the hepatopancreas of uninfected snails, whereas the Lewis X epitope was only weakly detectable in the latter tissue. In contrast, increased binding of antibodies recognizing LDN, LDN-F and LDN-DF was observed in infected snail tissue, in particular in regions involved in sporocystogenesis, in addition to an enhanced binding of anti-SEA serum and antibodies reacting with F-LDN. A pronounced expression of most of these carbohydrate antigens was also observed at the surface of miracidia. Hence, the detection of shared carbohydrate determinants in uninfected snail tissue, sporocysts and miracidia may support the hypothesis of carbohydrate-based molecular mimicry as a survival strategy of S. mansoni.     

Surface membrane proteins of Biomphalaria glabrata embryonic cells bind fucosyl determinants on the tegumental surface of Schistosoma mansoni primary sporocysts

Previous observations that in vitro adherence of Biomphalaria glabrata embryonic (Bge) cells to sporocyst larval stages of Schistosoma mansoni was strongly inhibited by fucoidan, a sulfated polymer of L-fucose, suggested a role for lectinlike Bge cell receptors in sporocyst binding interactions. In the present investigation, monoclonal antibodies with specificities to 3 major glycan determinants found on schistosomes, LacdiNAc, fucosylated LacdiNAc (LDNF), and the Lewis X antigen, were used in adhesion blocking studies to further analyze the molecular interactions at the host-parasite interface. Results showed that only the anti-LDNF antibody significantly reduced snail Bge cell adhesion to the surface of sporocysts, suggesting that fucosyl determinants may be important in larval-host cell interactions. Affinity chromatographic separation of fucosyl-reactive Bge cell proteins from fucoidan-bound Sepharose 4B revealed the presence of polypeptides ranging from 6 to 200 kDa after elution with fucoidan-containing buffer. Pre-elution of the Bge protein-bound affinity column with dextran (Dex) and dextran sulfate (DexS) before introduction of the fucoidan buffer served as controls for protein binding based on nonspecific sugar or negative charge interactions. A subset of polypeptides (approximately 35-150 kDa) released by fucoidan elution was identified as Bge surface membrane proteins, representing putative fucosyl-binding proteins. Far-western blot analysis also demonstrated binding reactivity between Bge cell and sporocyst tegumental proteins. The finding that several of these parasite-binding Bge cell proteins were also fucoidan-reactive suggests the possible involvement of these molecules in mediating cellular interactions with sporocyst tegumental carbohydrates. It is concluded that Bge cells have surface protein(s) that may be playing a role in facilitating host cell adhesion to the surface of schistosome primary sporocysts through larval fucosylated glycoconjugates.     

Lectin binding to cells of Schistosoma mansoni sporocysts and surrounding Biomphalaria glabrata tissue

The binding of different lectins to the surface of mother and daughter sporocysts of Schistosoma mansoni (Trematoda) and to cells of its intermediate host Biomphalaria glabrata (Gastropoda) was investigated. The test system consisted of several biotin-labeled lectins, an avidin-biotin-peroxidase complex and 3,3'-diaminobenzidine. The fixatives used were Formalin, Bouin's and Zenker's solutions; unfixed material was also studied. Most lectins reacted equally with host tissue and parasite tissue. However, receptors for Ulex europaeus I (most probably fucose) were only demonstrated on daughter sporocysts. Thus, a method was found to specifically mark Schistosoma mansoni daughter sporocysts in the digestive gland tissue of its intermediate host. Mother sporocysts and surrounding host tissue differed in their distribution of galactosyl groups. Both lack fucose and N-acetyl-galactosamine. The differences in lectin binding of galactosyl determinants were also observed during the in vitro development of mother to daughter sporocysts.     

Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts

Miracidia (and adults) of Schistosoma mansoni which had been subjected to particle bombardment with a plasmid DNA encoding enhanced green fluorescent protein (EGFP) under control of the S. mansoni heat shock protein 70 (HSP70) promoter and termination elements were shown to express the reporter gene. Bombarded miracidia were able to penetrate and establish in Biomphalaria glabrata the intermediate host snail. Gold particles could be detected in the germ balls of parasites in paraffin-sections of snail tissue. The bombarded miracidia were able to develop normally and to transform into mother sporocysts. Reporter gene activity could be determined at 10 days post-infection by RT-PCR in snail tissues, but not by microscopy or Western blot which probably reflected sub-optimal expression levels of constructs. Our findings indicated that it is feasible to return transgenic miracidia to the life cycle, a crucial step for the establishment of a transgenesis system for schistosomes.     

Development of Trichobilharzia australis Blair & Islam, 1983 in the snail, Lymnaea lessoni Deshayes and in an experimental definitive host, the Muscovy duck

The development of Trichobilharzia australis Blair & Islam, 1983 in the intermediate host, Lymnaea lessoni Deshayes and in experimental definitive hosts, Muscovy ducks is described. 24 hours after entry into the snail, miracidia had lost their cilia, epidermal plates and lateral processes and became young mother sporocysts. They were located in the tissues of the head-foot organ of the snail and were oval in cross section but still retained the shape of a miracidium, and measured 0.058-0.066 X 0.041-0.049 mm. From the seventh day onward young mother sporocysts were tubular, thin-walled, irregular in shape and were in the tissues of the lung and kidney of the snail. On the 24th day mature mother sporocysts and young daughter sporocysts were found in the digestive gland. Between the 24th and 29th day mature daughter sporocysts with fully developed cercariae ready to emerge, or already emerged, could be seen in the digestive gland of the snail. Cercariae emerged from the snail from the 29th to 46th day after exposure at 25 degrees +/- 1 degree C. The prepatent period of T. australis in the Muscovy ducks was 22 to 42 days after the beginning of exposure to cercariae.     

A potential vector of Schistosoma mansoni in Uruguay

Susceptibility experiments were carried out with a Biomphalaria straminea-like planorbid snail (Biomphalaria aff. straminea, species inquirenda) from Espinillar, near Salto (Uruguay), in the area of the Salto Grande reservoir, exposed individually to 5 miracidia of Schistosoma mansoni (SJ2 and BH2 strains). Of 130 snails exposed to the SJ2 strain, originally infective to Biomphalaria tenagophila, 30 became infected (23%). The prepatent (precercarial) period ranged from 35 to 65 days. The cercarial output was irregular, following no definite pattern, varying from 138 to 76,075 per snail (daily average 4.3 to 447.5) and ending up with death. Three specimens that died, without having shed cercariae, on days 69 (2) and 80 after exposure to miracidia, had developing secondary sporocysts in their tissues, justifying the prospect of a longer precercarial period in these cases. In a control group of 120 B. tenagophila, exposed to the SJ2 strain, 40 became infected, showing an infection rate (33.3%) not significantly different from that of the Espinillar snail (chi 2 = 3.26). No cercariae were produced by any of the Espinillar snails exposed to miracidia of the BH2 strain, originally infective to Biomphalaria glabrata. Four specimens showed each a primary sporocyst in one tentacle, which disappeared between 15 and 25 days post-exposure, and two others died with immature, very slender sporocysts in their tissues on days 36 and 54. In a control group of 100 B. glabrata exposed to BH2 miracidia, 94 shed cercariae (94%) and 6 remained negative. Calculation of Frandsen's (1979a, b) TCP/100 index shows that "Espinillar Biomphalaria-SJ2 S. mansoni" is a vector-parasite "compatible" combination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selection of genotypes of Schistosoma mansoni and their maintenance by sporocyst transplantation

Schistosoma mansoni was isolated by hatching eggs obtained from a naturally infected Rat in Grand Etang, Guadeloupe; fifty Biomphalaria glabrata were exposed to five miracidia each. The resulting cercariae were used to infect laboratory mice which were later sacrificed to provide worms for enzyme analyses and eggs for further infections. Seven enzymes in extracts of individual worms were examined by isoelectric focusing in polyacrylamide gels: AcP, G6PDH, PGM, GPI and HK showed no variation, whereas MDH and LDH proved to be polymorphic. Two MDH loci were recognised, MDH-2 was invariant whereas two alleles were assumed at the MDH-1 locus. It was not possible to make a genetic interpretation of the complex banding pattern of LDH, although 4 types (LDH-A, -B, -C, -D) were observed. Of the snail infections, one batch of snails was exposed to 5 miracidia per snail in the normal way whereas other snails were each exposed to a single miracidium. The latter were sacrificed to provide sporocysts to transplant into further groups of recipient snails. Cercariae from the recipient snails were used to infect mice and the adult worms were analysed and compared with the normally passaged material. In this way, three lines, defined by the possession of particular MDH and LDH types, were selected from the originally polymorphic population; two were identical. The combination of single miracidium infections and enzyme typing has illustrated the possibility of selecting parasite lines of known genotype; transplantation of sporocysts from snail to snail has demonstrated that such lines can be maintained exclusively in the intermediate host.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of amphotericin B on the infection success of Schistosoma mansoni in Biomphalaria glabrata

In the present study, we examined the effect of amphotericin B on larval stages (miracidia and primary sporocyst) of the helminth Schistosoma mansoni, the causative agent of human schistosomiasis. Amphotericin B (AmB) is a polyene macrolide that disturbs the function of the cell membrane; it is widely used as prophylactic antimycotic agent in in vitro culture. We show for the first time that S. mansoni miracidia infectivity is considerably reduced after AmB treatment. Moreover we demonstrate that AmB does not affect the development, growth, viability, and behavior of miracidia and primary sporocysts. Our data indicate that AmB effects on S. mansoni sporocyst prevalence are linked to the oxidative properties of AmB. These may alter the capacity of sporocysts to respond to the oxidative stress generated by the snail immune defence system.     

Sublethal effects of ultraviolet b radiation on miracidia and sporocysts of Schistosoma mansoni: intramolluscan development, infectivity, and photoreactivation

Schistosoma mansoni occurs in tropical regions where levels of ultraviolet B (UVB; 290-320 nm) light are elevated. However, the effects of UVB on parasite transmission are unknown. This study examines effects of UVB on the miracidia and sporocysts of S. mansoni, focusing specifically on intramolluscan development, infectivity, and the ability to photoreactivate (repair DNA damage using visible light). Histology revealed that miracidia irradiated with 861 J x m(-2) underwent abnormal development after penetrating Biomphalaria glabrata snails. Total number of sporocysts in snail tissues decreased as a function of time postinfection (PI), among both nonirradiated and irradiated parasites; however, this decrease was greater in the latter. Moreover, whereas the proportion alive of nonirradiated sporocysts increased PI, that of irradiated sporocysts, i.e., derived from irradiated miracidia, decreased. Irradiation of miracidia with UVB resulted in decreased prevalence of patent infection (defined by presence of daughter sporocysts) in a dose-dependent manner, and no infections occurred at a dose of 861 J x m(-2). Like many aquatic organisms, including the snail host, parasites were able to photoreactivate if exposed to visible light following UVB irradiation, even subsequent to penetrating snails. These photoreactivation results suggest cyclobutane-pyrimidine dimers in DNA as the primary mechanism of UVB damage, and implicate photoreactivation, rather than nucleotide excision, as the main repair process in S. mansoni.     

Lecithochirium furcolabiatum (Jones 1933), Dawes 1947: the miracidium and mother sporocyst.

Experimental infections of the marine topshell Gibbula umbilicalis with Lecithochirium furcolabiatum (Digenea: Hemiuroidea) have allowed the development of a model system which will enable further studies of the molluscan host response. The long-lived intertidal prosobranch host is easily maintained in the laboratory, and experimental infection rates of 98% were consistently achieved. The miracidium and mother sporocyst have been studied at both light and ultrastructural levels, providing the first account of the morphology of these stages in Hemiuridae. The ingested egg hatches within the host intestine, treatment with L-cysteine and alkaline pH stimulating miracidial emergence in vitro. The general body surface of the miracidium is devoid of spines or cilia, the latter being restricted to four plates near the anterior extremity. The miracidium swims actively prior to penetration of the gut wall, the sporocyst being released from the miracidial epidermal coat within the haemocoel. Within 5 weeks of infection, the filamentous mother sporocyst contains 1 to 3 oval germ balls, daughter sporocysts being recorded free within the digestive gland haemocoel 7 weeks later. Twenty three weeks after ingestion of eggs, the daughter sporocyst extends into the host gill filaments, containing cystophorous cercariae ready for emergence.     

Protein kinase C signalling during miracidium to mother sporocyst development in the helminth parasite, Schistosoma mansoni

For schistosomes, development of the miracidium to mother sporocyst within a compatible molluscan host requires considerable physiological and morphological changes by the parasite. The molecular mechanisms controlling such development have not been explored extensively. To begin to elucidate the importance of kinase-mediated signal transduction to this process, the phosphorylation (activation) of protein kinase C (PKC) in larval stages of Schistosoma mansoni undergoing in vitro transformation was explored. Mining of the S. mansoni genomic database revealed two S. mansoni PKC proteins with high homology to human PKCβ and containing the conserved autophosphorylation (activation) site represented by serine 660 of human PKCβ_II. Western blotting with anti-phosphospecific antibodies directed to this site demonstrated that miracidia freshly-hatched from eggs possessed PKC (78 kDa) which was phosphorylated (activated) when miracidia were exposed to phorbol ester, and dephosphorylated (inhibited) following exposure to the PKC inhibitor GF109203X. Miracidia treated with the phospholipase C (PLC) inhibitor U73122 also displayed decreased PKC phosphorylation. S. mansoni PKC was phosphorylated during the initial 24 h development of miracidia into mother sporocysts; after 31 h and 48 h development, phosphorylation was reduced by 72% and 86%, respectively. Confocal microscopy of miracidia revealed phosphorylated PKC associated with the neural mass, excretory vesicle, tegument, ciliated plates, terebratorium and germinal cells; in larvae undergoing transformation for 31 h, phosphorylated PKC was only occasionally detected, being present in regions likely corresponding to the ridge cyton. Inhibition of PKC in miracidia by GF109230X resulted in accelerated transformation, particularly to the postmiracidium stage; ciliated plates were also shed from developing larvae more rapidly. These results highlight the dynamic nature of PKC signalling during S. mansoni postembryonic development and support a role for active PKC in restricting transformation of S. mansoni miracidia into mother sporocysts.     

Host parasite relationship among 3 Schistosoma mansoni and 6 Biomphalaria glabrata strains from epidemic and no epidemic Venezuelan areas is studied

Six lots of 18 B. glabrata from: La Victoria, Turmero, Cagua in Aragua state; Caserío El 25 in Carabobo state, Chabasquén in Portuguesa state and Humocaro Bajo in Lara state, were experimentally infected with miracidia of SM, C5 and C6 strains of Schistosoma mansoni (18 snails/Schistosoma mansoni strain). The averages of the intramolluscal period (IMP) obtained for the S. mansoni strains were very similar and comprised between 35.4 and 36.1 days. No significative statistical differences in the IMP were found according to the S. mansoni strain and the size of snails: < 7 mm and > 7 mm. However, significative statistical differences in the IMP were found, in relation to the B. glabrata strain and between the snails classified in two groups according to the S. mansoni dose (5 miracidia/snail and 10 miracidia/snail). The higher percentages of infection (PI) were found for the following parasite-snail combinations: C6-Cas. El 25 (80.7%), SM-La Victoria (73.1%) and C5-Cagua (62%). No significative statistical differences were found for the PI a) between the snail classified in two groups according to the size (< 7 mm and > 7 mm), b) in relation to the miracidium dosification (5 and 10 miracidia/snail and c) in accord to the S. mansoni strain. However, significative statistical differences were found for the PI obtained with different strains of the snail.     

Serotonin-induced muscular activity in Schistosoma mansoni larval stages: importance of 5-HT transport and role in daughter sporocyst production

Mother sporocysts of Schistosoma mansoni transport exogenously supplied serotonin (5-hydroxytrypamine; 5-HT), and respond to it with increases in motility. In the present study, we investigated the importance of 5-HT transporter activity in the manifestation of these 5-HT-induced motility changes, and further examined the role of 5-HT in the development of daughter sporocysts in vitro. Serotonin-induced motility of in vitro-derived sporocysts is not inhibited by antidepressant compounds, e.g., fluoxetine, that block 5-HT transport, suggesting that the receptors responsible for motility responses to 5-HT are surface exposed. Using a sporocyst in vitro culture system, we show that depletion of larval stores of 5-HT reduces production of daughter sporocysts, the second intramolluscan larval stage. Moreover, we demonstrate a strong correlation between endogenous 5-HT levels and basal mother sporocyst muscle activity. Overall, these data suggest that larval stages of S. mansoni can detect exogenous 5-HT via surface-exposed receptors, and they are consistent with the hypothesis that endogenous stores of 5-HT are important for the proper regulation of muscular contractions in mother sporocysts, and for the successful emergence of daughter sporocysts.