أصول مستقلة وتوقيعات اختيار غير متوازية لمقاومة التريكلا بندازول في فاسيولا هيباتيكا

تم القبول: 4 مارس 2025

النتائج

تسلسل جينوم سكان الكبد البالغ لفاسيولا هيفاتيكا مع اختلاف في قابلية التريكلابندازول

لا تظهر تجمعات الفاسيولا في منطقة كوسكو في بيرو هيكلًا جينيًا كبيرًا فيما يتعلق بظاهرة حساسية TCBZ الخاصة بها.

قمنا بمقارنة عيناتنا البيروفية مع بيانات تسلسل الجينوم الفردي المنشورة سابقًا من المملكة المتحدة والولايات المتحدة وأوروغواي. ) لوضع التنوع الجيني في سياقه بين المناطق الجغرافية المتنوعة مجموعات .hepatica أشارت أنماط مشاركة الأليلات على مستوى الجينوم إلى أن تتميز مجموعة . hepatica في بيرو عن العينات المجمعة في المملكة المتحدة وأوروغواي، وهو ما يتماشى مع تدفق الجينات المحدود المتوقع بين هذه المواقع (الشكل 1ب). لوحظ مستوى أقل من التمايز الجيني بين العينات من بيرو وعينة من أوريغون، الولايات المتحدة الأمريكية، كما تم الإشارة إليه سابقًا. . من بين العينات البيروفية، كان هناك القليل من الهيكلة الجينية بين الديدان ذات الفينوتيب الحساس لتأثير TCBZ المتباين (الشكل 1c). من خلال مقارنة المسافات بين المجموعات وداخل المجموعة بناءً على الهوية حسب الحالة (IBS) SNPs المقطوعة بواسطة LD مع تردد أليل ثانوي )، سألنا عما إذا كانت، في المتوسط، الأزواج عبر المجموعتين أقل تشابهًا من الأزواج داخل مجموعة الظاهرة مما يمكن توقعه بالصدفة. تم الحصول على نتيجة اختبار غير دالة ( ; 91 عينة TCBZS و194 عينة TCBZ-R، المتوسط والانحراف المعياري لـ ، و ، لمجموعة IBS بين المجموعات، و IBS داخل المجموعة (TCBZ-S)، و IBS داخل المجموعة (TCBZ-R) على التوالي؛ اختبار التبديل الذي يقوم بتغيير تسميات الظاهرة، التباديل)، مما يشير إلى أن عيناتنا تم أخذها من مجموعة تزاوج محلية دون وجود تباين سكاني قوي بين ديدان TCBZ-S و -R. كان من الممكن أن يؤدي التباين إلى تشويش تحليل توقيعات الانتقاء. تقديرات التنوع النوكليوتيدي على مستوى الجينوم ( كانت تقديرات Tajima’s D المتوسطة على مستوى الجينوم إيجابية (>0.6) في كل من مجموعات TCBZ-S و -R، مما يشير إلى احتمال حدوث انكماش سكاني حديث في المنطقة.
F. hepatica هو خنثى بنظام تزاوج مختلط يتضمن كل من التزاوج الداخلي والتزاوج الخارجي. في الأنواع التي تتزاوج داخليًا، غالبًا ما تظهر المواقع المرتبطة ارتباطًا وثيقًا هيكلًا من الأنماط الوراثية يمكن اكتشافه كاختلال ارتباط مرتفع. في مجموعة الدراسة لدينا، تدهور الاختلال إلى قيمة بالنسبة لـ SNPs المفصولة بمقدار 20 كيلوبايت واقتربت من مستويات الخلفية عند مسافات أكبر من 250 كيلوبايت (الشكل 1f)، فإن النمط مشابه لتلك الموجودة في أنواع أخرى من التزاوج الذاتي ذات أنظمة تزاوج مختلطة. يغير التزاوج الداخلي ترددات الجينات من خلال زيادة التماثل الجيني، بشكل كبير في شكل سلاسل من التماثل الجيني (ROH)، مما يقلل من الفعالية

تشمل المواقع المرشحة تحت اختيار TCBZ EGFR/PI3K-mTOR-

تحليل ملفات النسخ حدد اختلاف التعبير في الجينات المرتبطة بالميكروتوبولات في الديدان TCBZ-S و -R

لفهم الفروقات الأساسية في التعبير الجيني بين الديدان TCBZ-S و -R وللتحقيق في كيفية اختلاف استجابة الأدوية بين الديدان ذات الأنماط الظاهرية المختلفة، قمنا بتوصيف ملفات التعبير الجيني للطفيليات البالغة المستمدة من الديدان الأبوية ذات الحساسية المعروفة للأدوية (الشكل التوضيحي 4 والبيانات التكميلية 6). تم إجراء تسلسل RNA على عينات الديدان الكاملة المعالجة وغير المعالجة بالأدوية، مع التركيز على (1) الفروقات في التعبير بين الديدان TCBZ-S و -R بدون علاج و (2) تأثيرات علاج TCBZ داخل كل مجموعة نمط ظاهري. لضمان نتائج إحصائية قوية، أخذنا في الاعتبار التباين المرتبط بالوقت و

في الديدان الطفيلية TCBZ-S، أظهرت 62 جينًا زيادة ملحوظة، وأظهرت 131 جينًا انخفاضًا ملحوظًا استجابةً لعلاج TCBZ؛ ومع ذلك، لم يتم الكشف عن أي جينات معبرة بشكل مختلف بشكل ملحوظ في الديدان الطفيلية TCBZ-R. و تغير الطي؛ الشكل 3ب، ج). كان الجين الأكثر زيادة بشكل ملحوظ هو كيناز البروتين المنشط بالميتوجين (المماثل للبشر MAPK11) ( )، التي تنتمي إلى فئة من الكينازات (p38 MAPKs) التي تستجيب لمحفزات الإجهاد وتشارك في تمايز الخلايا، وموت الخلايا المبرمج، والالتهام الذاتي (الجدول 2). لم نلاحظ أي فئات وظيفية غنية بشكل ملحوظ ( ) من بين 62 جينًا مرتفع التعبير (الشكل 3e). الـ 131

يمكن تمييز طفيليات TCBZ-S و -R باستخدام عدد محدود من SNPs المعلوماتية

الشكل 4 | تمييز فاسيولا الحساسة والمقاومة للتركلابندازول
مستندة إلى ملفات SNP. تصنيف المجموعات بواسطة تحليل التمييز للمكونات الرئيسية (DAPC). تم استخدام أفضل 300 SNP، التي تظهر اختلافات ملحوظة في تردد الأليلات بين المجموعات وليست في توازن ربط قوي مع بعضها البعض، لتصنيف 91 عينة WGS من TCBZ-S و194 عينة من TCBZ-R.

b

نقاش

طرق

الحصول على الطفيليات وتسلسل الجينوم الكامل

تحليلات مسح المتغيرات والاختيار على مستوى الجينوم

توليد TCBZ-S و -R ميتا سيركاريا

توليد طفيليات TCBZ-S و -R البالغة . هيفاتيكا

تجارب التعرض لـ Triclabendazole وتحليل التعبير الجيني باستخدام RNA-seq

تصميم لوحة الأمبليكون والتسلسل المستهدف (amplicon-seq)

تمييز نمط حساسية TCBZ باستخدام مجموعة من علامات SNP

ملخص التقرير

توفر البيانات

توفر الشيفرة

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شكر وتقدير

مساهمات المؤلفين

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معلومات إضافية

معلومات إضافية النسخة الإلكترونية تحتوي على

Independent origins and non-parallel selection signatures of triclabendazole resistance in Fasciola hepatica

Accepted: 4 March 2025

Results

Population sequencing of adult Fasciola hepatica with divergent triclabendazole susceptibility

Fasciola populations in the Cusco region of Peru do not exhibit significant genetic structuring with respect to their TCBZ sensitivity phenotype

We compared our Peruvian samples to previously published individual genome sequencing data from the UK, US, and Uruguay ( ) to contextualize genetic variation among geographically diverse . hepatica populations . Genome-wide allele sharing patterns indicated that the . hepatica population in Peru is distinct from specimens collected in the UK and Uruguay, consistent with the expected limited gene flow between these locations (Fig. 1b). A lower level of genetic differentiation was observed between samples from Peru and an isolate from Oregon, USA, as previously noted . Among the Peruvian samples, there was little genetic structuring between flukes with divergent TCBZ sensitivity phenotype (Fig. 1c). By comparing betweengroup and in-group pairwise identity-by-state (IBS) distances (based on LD-pruned SNPs with a minor allele frequency ), we asked whether, on average, pairs across the two groups are less similar than pairs within a phenotype group than would be expected by chance. A non-significant test result was obtained ( ; 91 TCBZS and 194 TCBZ-R samples, mean and standard deviation of , and , for betweengroup IBS, in-group (TCBZ-S) IBS, and in-group (TCBZ-R) IBS, respectively; permutation test randomizing phenotype labels, permutations), suggesting that our specimens were sampled from a local interbreeding population without strong population stratification between TCBZ-S and -R flukes. Stratification could have confounded the analysis of selection signatures. Genome-wide estimates of nucleotide diversity ( ) were 0.0065 regardless of TCBZ sensitivity (Fig. 1d). Genome-wide mean Tajima’s D estimates were positive (>0.6) in both TCBZ-S and -R populations (Fig. 1e), suggesting a possible recent population contraction in the region.
F. hepatica is a hermaphrodite with a mixed mating system involving both inbreeding and outcrossing. In inbreeders, closely linked sites often show haplotype structure detectable as high linkage disequilibrium (LD). In our study population, LD decayed to a value of for SNPs separated by 20 kb and approached background levels at distances greater than 250 kb (Fig. 1f), a pattern comparable to those in other selfing species with mixed mating systems . Inbreeding changes genotype frequencies by increasing homozygosity, largely in the form of runs of homozygosity (ROH), thereby reducing effective

Candidate loci under TCBZ selection include EGFR/PI3K-mTOR-