Comparison of Echinostoma caproni Mother Sporocyst Development In vivo and In vitro Using Biomphalaria glabrata Snails and a B. glabrata Embryonic Cell Line

Abstract Background: Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro.Methods: Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed.Results: Gene knockout manipulation induced gene-disrupting indels, frequently 1–2 bp insertions and/or 8–30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n =12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01).Conclusions: The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

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Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

10.21203/rs.3.rs-51130/v2 ◽

2020 ◽

Author(s):

Fernanda Sales Coelho ◽

Rutchanee Rodpai ◽

Andre Miller ◽

Shannon E Karinshak ◽

Victoria H Mann ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Cell Line ◽

Biomphalaria Glabrata ◽

Embryonic Cell ◽

Gene Transcript ◽

Inflammatory Factor ◽

Wild Type ◽

Transcript Levels ◽

Embryonic Cell Line

Abstract Background: Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro.Methods: Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by quantitative reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed.Results: Gene knockout manipulation induced gene-disrupting indels, frequently 1-2 bp insertions and/or 8-30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5±20.2% S.D, P ≤ 0.05, n =12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66±0.10, ΔBgAIF, 2.30±0.22 (P ≤ 0.01).Conclusion: The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

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Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

Parasites & Vectors ◽

10.1186/s13071-020-04384-9 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Fernanda Sales Coelho ◽

Rutchanee Rodpai ◽

André Miller ◽

Shannon E. Karinshak ◽

Victoria H. Mann ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Cell Line ◽

Biomphalaria Glabrata ◽

Embryonic Cell ◽

Gene Transcript ◽

Inflammatory Factor ◽

Wild Type ◽

Transcript Levels ◽

Embryonic Cell Line

Abstract Background Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro. Methods Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed. Results Gene knockout manipulation induced gene-disrupting indels, frequently 1–2 bp insertions and/or 8–30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n = 12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01). Conclusions The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

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Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

10.1101/2020.04.07.029629 ◽

2020 ◽

Author(s):

Fernanda Sales Coelho ◽

Rutchanee Rodpai ◽

André Miller ◽

Shannon E. Karinshak ◽

Victoria H. Mann ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Cell Line ◽

Biomphalaria Glabrata ◽

Embryonic Cell ◽

Gene Transcript ◽

Inflammatory Factor ◽

Wild Type ◽

Transcript Levels ◽

Embryonic Cell Line

AbstractBackgroundLarval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro.MethodsBge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by quantitative reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed.ResultsGene knockout manipulation induced gene-disrupting indels, frequently 1-2 bp insertions and/or 8-30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5±20.2% S.D, P ≤ 0.05, n =12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66±0.10, ΔBgAIF, 2.30±0.22 (P ≤ 0.01).ConclusionThe findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

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Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick—Pathogen Interactions

Pathogens ◽

10.3390/pathogens10010070 ◽

2021 ◽

Vol 10 (1) ◽

pp. 70

Author(s):

Lourdes Mateos-Hernández ◽

Natália Pipová ◽

Eléonore Allain ◽

Céline Henry ◽

Clotilde Rouxel ◽

...

Keyword(s):

Cell Line ◽

Peripheral Nerves ◽

Ixodes Scapularis ◽

Embryonic Cell ◽

Cell Subpopulation ◽

In Vivo Experiments

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick–pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick–pathogen interactions.

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Cercarial tail loss in Echinostoma caproni: the influence of in vivo encystment and copper sulphate

Journal of Helminthology ◽

10.1079/joh2005278 ◽

2005 ◽

Vol 79 (2) ◽

pp. 177-178 ◽

Cited By ~ 1

Author(s):

B. Fried ◽

J.L. Schneck

Keyword(s):

Copper Sulphate ◽

Biomphalaria Glabrata ◽

Spring Water ◽

Echinostoma Caproni ◽

Tail Loss ◽

Water Ph

AbstractEchinostoma caproni tail loss was studied in vitro in the presence of the toxicant copper sulphate (CuSO4) in concentrations ranging from 1 to 10 000 mg l?1 in standardized artificial spring water (pH 7.4, osmolarity 34 mOsm kg?1 H2O, Ca2+ 20 mg l?1) at 23°C. Tail loss was also studied in the absence of toxicants during in vivo encystment of the cercariae in juvenile Biomphalaria glabrata. As the concentration of CuSO4 increased, the percentage of cercarial tail loss increased. By 2 h in 10 000 mg l?1, 1000 mg l?1 and 100 mg l?1 CuSO4, 50%, 23% and 13%, respectively, of the cercariae had lost their tails. In the in vivo studies, by 1 h PI, 59±5% of cercariae had lost their tails and only 4±1% of the cercariae were actively swimming in the multi-well dishes. At 3 h PI, 72±3% of the cercariae began to form cysts within the snails.

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