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Liquid biopsy (LB) ra'ayi ne da ke samun karbuwa cikin sauri a fagen nazarin halittu.Manufar ta dogara ne akan gano gutsuttsura na DNA na extracellular (ccfDNA), waɗanda galibi ana fitar da su azaman ƙananan gutsuttsura bayan mutuwar tantanin halitta a cikin kyallen takarda daban-daban.Kadan daga cikin waɗannan guntuwar sun samo asali ne daga kyamarorin halitta ko na waje (baƙi).A cikin aikin na yanzu, mun yi amfani da wannan ra'ayi ga mussels, nau'in simintin da aka sani da ƙarfin tace ruwan teku.Muna amfani da ikon mussels don aiki azaman masu tacewa na halitta don ɗaukar gutsuttsuran DNA na muhalli daga tushe iri-iri don samar da bayanai game da bambancin halittun halittun tekun teku.Sakamakon mu ya nuna cewa mussel hemolymph ya ƙunshi guntun DNA waɗanda suka bambanta da girma, daga 1 zuwa 5 kb.Tsarin harbin bindiga ya nuna cewa ɗimbin guntuwar DNA na asalin ƙananan ƙwayoyin cuta ne na ƙasashen waje.Daga cikin su, mun sami gutsuttsarin DNA daga ƙwayoyin cuta, archaea, da ƙwayoyin cuta, gami da ƙwayoyin cuta da aka sani don cutar da runduna iri-iri da aka fi samu a cikin yanayin yanayin tekun teku.A ƙarshe, bincikenmu ya nuna cewa manufar LB da aka yi amfani da ita ga mussels tana wakiltar wadata amma har yanzu tushen ilimin da ba a gano ba game da bambancin ƙananan ƙwayoyin cuta a cikin yanayin tekun teku.
Tasirin sauyin yanayi (CC) akan halittun halittun ruwa wani yanki ne na bincike cikin sauri.Dumamar duniya ba wai kawai yana haifar da mahimman matsalolin physiological ba, har ma yana tura iyakokin juyin halitta na kwanciyar hankali na thermal na halittun ruwa, yana shafar mazaunan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan da nau'ikan ke haifar da haɓaka iyakoki na daidaita yanayin yanayin yanayin yanayin yanayin yanayin yanayin yanayin yanayin yanayin yanayin yanayi [1,2].Bugu da ƙari, yana shafar bambancin halittu na metazoans, CC yana rushe ma'auni mai laushi na hulɗar mahalli-microbial.Wannan microbial dysbacteriosis yana haifar da mummunar barazana ga yanayin halittu na ruwa yayin da yake sa kwayoyin ruwa su fi dacewa da cututtuka masu cututtuka [3, 4].An yi imanin cewa SS na taka muhimmiyar rawa wajen yawan mace-mace, wanda babbar matsala ce ga kula da yanayin yanayin ruwa na duniya [5, 6].Wannan lamari ne mai mahimmanci idan aka yi la'akari da tasirin tattalin arziki, muhalli da abinci mai gina jiki na yawancin nau'in ruwa.Wannan gaskiya ne musamman ga bivalves da ke zaune a cikin yankuna na polar, inda tasirin CK ya fi gaggawa kuma mai tsanani [6, 7].A zahiri, bivalves kamar Mytilus spp.ana amfani da su sosai don saka idanu akan tasirin CC akan yanayin yanayin ruwa.Ba abin mamaki ba ne, an ɓullo da adadi mai yawa na masu nazarin halittu don saka idanu akan lafiyar su, sau da yawa ta yin amfani da tsarin matakai biyu wanda ya haɗa da masu aikin biomarkers dangane da ayyukan enzymatic ko ayyukan salula kamar ƙarfin tantanin halitta da aikin phagocytic [8].Waɗannan hanyoyin kuma sun haɗa da auna ma'auni na ƙayyadaddun alamun matsa lamba waɗanda ke taruwa a cikin kyallen takarda masu laushi bayan sha mai yawa na ruwan teku.Koyaya, babban ƙarfin tacewa da tsarin siginar buɗe ido na bivalves suna ba da dama don haɓaka sabbin ƙwayoyin haemolymph ta amfani da manufar biopsy na ruwa (LB), hanya mai sauƙi da ƙarancin ɓarna ga kulawar haƙuri.samfurin jini [9, 10].Ko da yake ana iya samun nau'ikan kwayoyin halitta da yawa a cikin LB na ɗan adam, wannan ra'ayi ya samo asali ne akan binciken jerin DNA na gutsuttsuran DNA na extracellular (ccfDNA) a cikin jini.A gaskiya ma, kasancewar DNA da ke yawo a cikin plasma na ɗan adam an san shi tun tsakiyar karni na 20 [11], amma a cikin 'yan shekarun nan ne kawai zuwan hanyoyin da aka yi amfani da su sosai ya haifar da ganewar asibiti bisa ccfDNA.Kasancewar waɗannan gutsutsayen DNA da ke yawo ya samo asali ne daga wani bangare na sakin kwayoyin halittar DNA (nukiliya da mitochondrial) bayan mutuwar tantanin halitta. A cikin mutane masu lafiya, ƙaddamar da ccfDNA yawanci yana da ƙasa (<10 ng/mL) amma ana iya haɓaka ta sau 5-10 a cikin marasa lafiya da ke fama da cututtuka daban-daban ko kuma suna fuskantar damuwa, wanda ke haifar da lalacewar nama. A cikin mutane masu lafiya, ƙaddamar da ccfDNA yawanci yana da ƙasa (<10 ng/mL) amma ana iya haɓaka ta sau 5-10 a cikin marasa lafiya da ke fama da cututtuka daban-daban ko kuma suna fuskantar damuwa, wanda ke haifar da lalacewar nama. У здоровых людей концентрация в ккДНК в норме низкая (<10 нг/мл), но может повышаться в 5–10 разин ологией или подвергающихся стрессу, приводящему к повреждению тканей. A cikin mutane masu lafiya, ƙaddamarwar cccDNA yawanci yana da ƙasa (<10 ng/mL), amma yana iya ƙaruwa sau 5-10 a cikin marasa lafiya tare da cututtuka daban-daban ko kuma ƙarƙashin damuwa wanda ke haifar da lalacewar nama.在健康个体中，ccfDNA 的浓度通常较低（<10 ng/mL）。从而导致组织损伤。在 健康 个体 中 ， ccfdna 的 浓度 较 低 （<10 ng/ml） 在 各 种5-10 从而 组织。 损伤 损伤 损伤Концентрации ccfDNA обычно низкие (<10 нг/мл) тологиями или стрессом, что приводит к повреждению тканей. Matsakaicin ccfDNA yawanci yana da ƙasa (<10 ng/ml) a cikin mutane masu lafiya, amma ana iya ƙara ninki 5-10 a cikin marasa lafiya tare da cututtuka daban-daban ko damuwa, yana haifar da lalacewar nama.Girman ɓangarorin ccfDNA ya bambanta sosai, amma yawanci jeri daga 150 zuwa 200 bp.[12].Ana iya amfani da nazarin ccfDNA da aka samu da kansa, watau, ccfDNA daga al'ada ko canza ƙwayoyin ƙwayoyin cuta, don gano kwayoyin halitta da canje-canje na epigenetic da ke cikin kwayoyin halitta da / ko mitochondrial genome, don haka taimakawa likitocin su zaɓi takamaiman hanyoyin kwantar da hankali na kwayoyin halitta [13].Koyaya, ana iya samun ccfDNA daga majiyoyin waje kamar ccfDNA daga ƙwayoyin tayi a lokacin daukar ciki ko daga gabobin da aka dasa [14,15,16,17].ccfDNA kuma muhimmin tushen bayanai ne don gano kasancewar acid nucleic na wakili mai kamuwa da cuta (baƙin waje), wanda ke ba da damar gano cututtukan da ba a iya kamuwa da su ba ta hanyar al'adun jini, da guje wa ɓarna biopsy na ƙwayar cuta [18].Lallai binciken da aka yi kwanan nan ya nuna cewa jinin ɗan adam ya ƙunshi ɗimbin tushen bayanai waɗanda za a iya amfani da su don gano ƙwayoyin cuta na ƙwayoyin cuta da ƙwayoyin cuta, kuma kusan 1% na ccfDNA da aka samu a cikin plasma na ɗan adam asalinsu daga ƙasashen waje ne [19].Waɗannan binciken sun nuna cewa ana iya tantance bambancin halittu na ƙwayoyin cuta masu yawo da ƙwayoyin cuta ta hanyar yin amfani da bincike na ccfDNA.Duk da haka, har zuwa kwanan nan, an yi amfani da wannan ra'ayi ne kawai a cikin mutane kuma, a ɗan ƙarami, a cikin wasu vertebrates [20, 21].
A cikin takarda na yanzu, muna amfani da yuwuwar LB don nazarin ccfDNA na Aulacomya atra, wani nau'in kudanci da aka fi samu a cikin tsibirin Kerguelen na subantarctic, rukuni na tsibiran da ke saman wani babban tudu wanda ya kafa shekaru miliyan 35 da suka wuce.aman wuta.Yin amfani da tsarin gwaji na in vitro, mun gano cewa gutsuttssun DNA a cikin ruwan teku ana ɗaukar su da sauri ta hanyar mussels kuma su shiga sashin hemolymph.Tsarin Shotgun ya nuna cewa mussel hemolymph ccfDNA yana ƙunshe da gutsuttsuran DNA na asalinsa da wanda ba nasa ba, gami da ƙwayoyin cuta na sinadirai da gutsuttsuran DNA daga halittun halittu masu kama da yanayin yanayin yanayin teku na sanyi mai aman wuta.Hemolymph ccfDNA kuma ya ƙunshi jerin ƙwayoyin cuta waɗanda aka samo daga ƙwayoyin cuta tare da jeri daban-daban.Mun kuma sami gutsuttsuran DNA daga dabbobi masu yawa kamar kifin kashi, anemones na teku, algae da kwari.A ƙarshe, bincikenmu ya nuna cewa za a iya samun nasarar amfani da ra'ayi na LB a kan invertebrates na ruwa don samar da wadataccen tarihin kwayoyin halitta a cikin yanayin ruwa.
Manya (tsawon 55-70 mm) Mytilus platensis (M. platensis) da Aulacomya atra (A. atra) an tattara su daga gaɓar duwatsu masu tsaka-tsaki na Port-au-France (049 ° 21.235 S, 070 ° 13.490 E .).Tsibirin Kerguelen a watan Disamba na 2018. Sauran manyan mussels blue (Mytilus spp.) An samo su daga mai siyar da kasuwanci (PEI Mussel King Inc., Prince Edward Island, Kanada) kuma an sanya shi a cikin yanayin zafi mai sarrafawa (4 ° C) wanda ke dauke da 10-20 L na 32 ‰ brine artificial.(Gishirin teku na wucin gadi Reef Crystal, Instant Ocean, Virginia, Amurka).Ga kowane gwaji, an auna tsayi da nauyin kwasfa ɗaya.
Ana samun ƙa'idar buɗe hanyar shiga kyauta don wannan shirin akan layi (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1).A taƙaice, an tattara LB hemolymph daga tsokoki masu sace kamar yadda aka bayyana [22].An bayyana hemolymph ta centrifugation a 1200 × g na minti 3, an daskarar da supernatant (-20 ° C) har sai an yi amfani da shi.Don keɓewa da tsarkakewar cfDNA, samfuran (1.5-2.0 ml) an narke kuma an sarrafa su ta amfani da kit ɗin NucleoSnap cfDNA (Macherey-Nagel, Bethlehen, PA) bisa ga umarnin masana'anta.ccfDNA an adana shi a -80°C har sai an ƙara bincike.A wasu gwaje-gwaje, an ware ccfDNA kuma an tsarkake ta ta amfani da Kit ɗin Binciken DNA na QIAamp (QIAGEN, Toronto, Ontario, Kanada).An ƙididdige DNA da aka tsarkake ta amfani da ma'auni na PicoGreen.Rarraba ɓangarorin ccfDNA keɓe an bincika ta hanyar electrophoresis na capillary ta amfani da Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) ta amfani da Babban Sensitivity DNA Kit.Anyi gwajin ta amfani da 1µl na samfurin ccfDNA bisa ga umarnin masana'anta.
Don jera guntuwar hemolymph ccfDNA, Génome Québec (Montreal, Quebec, Kanada) sun shirya ɗakunan karatu na harbi ta amfani da Illumina DNA Mix kit na Illumina MiSeq PE75 kit.An yi amfani da adaftar ma'auni (BioO).Fayilolin bayanai na asali suna samuwa daga Taskar Karatun Jeri na NCBI (SRR8924808 da SRR8924809).An kimanta ingancin karatun asali ta amfani da FastQC [23].An yi amfani da trimmomatic [24] don yankan adaftan da ƙarancin karantawa.An haɗa FLASH zuwa karantawa guda ɗaya mafi tsayi tare da mafi ƙarancin zoba na 20 bp don guje wa rashin daidaituwa [25]. An ba da bayanin haɗaɗɗen karatun tare da BLASTN ta amfani da bayanan NCBI Taxonomy bivalve (e ƙimar <1e-3 da 90% homology), kuma an yi amfani da DUST [26]. An ba da bayanin haɗaɗɗen karatun tare da BLASTN ta amfani da bayanan NCBI Taxonomy bivalve (e ƙimar <1e-3 da 90% homology), kuma an yi amfani da DUST [26]. Объединенные чтения были аннотированы с помощью BLASTN смотреть начение e <1e-3 и 90% гомологии), а маскирование последовательностей низкой An ba da bayanin karatun da aka karanta tare da BLASTN ta yin amfani da bayanan taxonomy na NCBI bivalve (e darajar <1e-3 da 90% homology), kuma an yi amfani da DUST [26].使用双壳类NCBI 分类数据库（e 值< 1e-3 和90%复杂度序列的掩蔽。使用 双 壳类 ncbi 分类 （（（<1e-3 和 90% 同源）复杂度 序列 的。。。 掩蔽 掩蔽 掩蔽蔽Объединенные чтения были аннотированы с помощью BLASTN с использованием NCBI (значение e <1e-3 и 90% гомологии), а маскирование An ba da bayanin karatun da aka karanta tare da BLASTN ta amfani da bayanan taxonomic na NCBI bivalve (e darajar <1e-3 da 90% homology), kuma an yi amfani da DUST [26].An raba karatun zuwa rukuni biyu: masu alaƙa da jerin bivalve (a nan ana kiran kai-karanta) da mara alaƙa (mara karatu).An haɗa ƙungiyoyi biyu daban ta amfani da MEGAHIT don ƙirƙirar contigs [27].A halin yanzu, an rarraba rarraba taxonomic na karatun microbiome na baƙi ta amfani da Kraken2 [28] kuma ginshiƙi na Krona ke wakilta ta hoto akan Galaxy [29, 30].An ƙaddara mafi kyawun kmers su zama kmers-59 daga gwajin mu na farko. Sannan an gano ƙididdiga na kai ta hanyar daidaitawa tare da BLASTN (bivalve NCBI database, e value <1e-10 da 60% homology) don bayanin ƙarshe. Sannan an gano ƙididdiga na kai ta hanyar daidaitawa tare da BLASTN (bivalve NCBI database, e value <1e-10 da 60% homology) don bayanin ƙarshe. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN 1e-10 и гомология 60%) для окончательной аннотации. Sannan an gano abubuwan da suka dace ta hanyar daidaitawa da BLASTN (NCBI bivalve database, e value <1e-10 da 60% homology) don bayanin ƙarshe.然后通过与 BLASTN（双壳贝类NCBI 数据库， e 值< 1e-10 和60%终注释.然后通过与 BLASTN（双壳贝类NCBI 数据库， e 值< 1e-10 和60% Затем были идентифицированы скачать видео - вустворчатых моллюсков, значение e <1e-10 и гомология 60%). Daga nan an gano na'urorin kai-da-kai don bayanin ƙarshe ta hanyar daidaitawa da BLASTN (NCBI bivalve database, e value <1e-10 da 60% homology). A cikin layi daya, an ba da bayanin ƙungiyoyin sa kai tare da BLASTN (n NCBI database, e ƙimar <1e-10 da 60% homology). A cikin layi daya, an ba da bayanin ƙungiyoyin sa kai tare da BLASTN (n NCBI database, e ƙimar <1e-10 da 60% homology). Параллельно чужеродные групповые контиги были аннотированы с помощью BLASTN . A cikin layi daya, an ba da bayanin ƙungiyoyin ƙasashen waje tare da BLASTN (Tsarin bayanan NT NCBI, ƙimar e <1e-10 da 60% homology).平行地，用BLASTN（nt NCBI 数据库，e 值< 1e-10 和60% 同源性）注释非自身组重叠群。平行地，用BLASTN（nt NCBI 数据库，e 值< 1e-10 和60% 同源性）注释非自身组重叠群。 Параллельно контиги, не относящиеся к собственной группе, были аннотированы с помощью BLASTN. 0 da гомология 60%). A layi daya, ƙungiyoyin da ba na kai ba an bayyana su tare da BLASTN (nt NCBI database, e value <1e-10 and 60% homology). Hakanan an gudanar da BLASTX akan abubuwan da ba na kai ba ta amfani da bayanan nr da RefSeq sunadaran NCBI (ƙimar e <1e-10 da 60% homology). Hakanan an gudanar da BLASTX akan abubuwan da ba na kai ba ta amfani da bayanan nr da RefSeq sunadaran NCBI (ƙimar e <1e-10 da 60% homology). BLASTX был проведен на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI0 60%). Hakanan an yi BLASTX akan abubuwan da ba na kai ba ta amfani da bayanan furotin na nr da RefSeq NCBI (darajar <1e-10 da 60% homology).还使用nr 和RefSeq 蛋白NCBI 数据库对非自身重叠群进行了BLASTX（e 值< 1e-10 和60% 吧还使用nr 和RefSeq 蛋白NCBI 数据库对非自身重叠群进行了BLASTX（e 值< 1e-10 和60% 吧 BLASTX также выполняли на несамостоятельных контигах с использованием баз данных белкя nr и RefSeq NCBI . Hakanan an yi BLASTX akan abubuwan da ba na kai ba ta amfani da bayanan furotin na nr da RefSeq NCBI (ƙimar e <1e-10 da 60% homology).Tafkunan BLASTN da BLASTX na abubuwan da ba na kai ba suna wakiltar ƙungiyoyin ƙarshe (duba fayil ɗin Ƙarin).
An jera abubuwan da aka yi amfani da su don PCR a cikin Table S1.An yi amfani da Taq DNA polymerase (Bio Basic Canada, Markham, ON) don haɓaka ƙwayoyin halittar ccfDNA.An yi amfani da yanayin halayen masu zuwa: denaturation a 95 ° C na minti 3, 95 ° C na minti 1, saita zafin jiki na 1 minti, elongation a 72 ° C na minti 1, hawan keke 35, kuma a ƙarshe 72 ° C cikin minti 10..An raba samfuran PCR ta hanyar electrophoresis a cikin gels agarose (1.5%) dauke da SYBRTM Safe DNA Gel Stain (Invitrogen, Burlington, ON, Kanada) a 95 V.
Mussels (Mytilus spp.) An ƙaddamar da su a cikin 500 ml na ruwan teku na oxygenated (32 PSU) na 24 hours a 4 ° C.DNA na Plasmid wanda ke ɗauke da abun da ke ɓoye jerin galectin-7 cDNA ɗan adam (lambar shiga L07769) an ƙara shi zuwa vial a matakin ƙarshe na 190 μg/μl.Mussels da aka haifa a ƙarƙashin yanayi iri ɗaya ba tare da ƙarin DNA ba sune sarrafawa.Tankin sarrafawa na uku ya ƙunshi DNA ba tare da mussels ba.Don saka idanu akan ingancin DNA a cikin ruwan teku, ana ɗaukar samfuran ruwan teku (20 μl; maimaitawa uku) daga kowane tanki a lokacin da aka nuna.Don gano DNA na plasmid, an girbe mussels na LB a lokutan da aka nuna kuma qPCR da ddPCR sun bincikar su.Saboda yawan gishirin ruwan teku, an diluted aliquots a cikin ingantaccen ruwa na PCR (1:10) kafin duk gwajin PCR.
An yi PCR dijital (ddPCR) ta amfani da ka'idar BioRad QX200 (Mississauga, Ontario, Canada).Yi amfani da bayanin martabar zafin jiki don tantance mafi kyawun zafin jiki (Table S1).An samar da digo ta amfani da janareta na QX200 (BioRad).ddPCR da aka gudanar kamar haka: 95°C na 5 min, 50 hawan keke na 95°C na 30 s da kuma ba annealing zafin jiki na 1 min da 72°C na 30 s, 4°C na 5 min da 90°C cikin minti 5.Adadin digo da ingantattun halayen (yawan kwafi/µl) an auna su ta amfani da mai karanta juzu'i na QX200 (BioRad).Samfurori masu ƙasa da ɗigo 10,000 an ƙi.Ba a aiwatar da tsarin sarrafawa duk lokacin da aka gudanar da ddPCR.
An yi qPCR ta amfani da Rotor-Gene® 3000 (Binciken Corbett, Sydney, Ostiraliya) da LGALS7 ƙayyadaddun firamare.Dukkan PCR masu ƙididdigewa an yi su a cikin 20µl ta amfani da QuantiFast SYBR Green PCR Kit (QIAGEN).An fara qPCR tare da shiryawa na 15 min a 95 ° C tare da zagayowar 40 a 95 ° C na daƙiƙa 10 kuma a 60 ° C na daƙiƙa 60 tare da tarin bayanai guda ɗaya.An ƙirƙiri maƙallan narkewa ta amfani da ma'auni masu zuwa a 95°C na 5s, 65°C don 60s, da 97°C a ƙarshen qPCR.An yi kowane qPCR a cikin sau uku, ban da samfuran sarrafawa.
Tun da an san mazugi da yawan tacewa, da farko mun bincika ko za su iya tacewa da riƙe gutsuwar DNA da ke cikin ruwan teku.Mun kuma yi sha'awar ko waɗannan guntuwar sun taru a cikin tsarin su na lymphatic.Mun warware wannan batu ta gwaji ta hanyar gano makomar gutsuttsuran DNA masu narkewa da aka saka a cikin tankuna masu shuɗi.Don sauƙaƙe bibiyar gutsuwar DNA, mun yi amfani da DNA na plasmid na waje (ba kai ba) mai ɗauke da kwayar halittar galectin-7 na ɗan adam.ddPCR yana gano gutsuttsuran DNA na plasmid a cikin ruwan teku da mussels.Sakamakonmu ya nuna cewa idan adadin gutsuttsuran DNA a cikin ruwan teku ya kasance mai ɗanɗano a tsawon lokaci (har zuwa kwanaki 7) in babu mussels, to a gaban mussels wannan matakin kusan ya ɓace gaba ɗaya cikin sa'o'i 8 (Fig. 1a,b).An gano gutsuttsuran DNA na waje a cikin sauƙi a cikin 15 min a cikin ruwan intravalvular da hemolymph (Fig. 1c).Ana iya gano waɗannan gutsuttsura har zuwa awanni 4 bayan fallasa.Wannan aikin tacewa dangane da gutsuttsuran DNA yana kwatankwacin aikin tacewa na kwayoyin cuta da algae [31].Wadannan sakamakon sun nuna cewa mussels na iya tacewa da tara DNA na waje a cikin sassan ruwa.
Matsakaicin dangi na plasmid DNA a cikin ruwan teku a gaban (A) ko rashi (B) na mussels, wanda aka auna ta ddPCR.A cikin A, ana bayyana sakamakon a matsayin kaso, tare da iyakokin kwalayen da ke wakiltar kashi 75 da 25.Ana nuna madaidaicin lanƙwan logarithmic da ja, kuma wurin da aka inuwa cikin launin toka yana wakiltar tazarar amincewa 95%.A cikin B, layin ja yana wakiltar ma'ana kuma layin shuɗi yana wakiltar 95% tazarar amincewa ga taro.C Tarin DNA plasmid a cikin hemolymph da valvular ruwa na mussels a lokuta daban-daban bayan ƙari na plasmid DNA.Ana gabatar da sakamakon azaman cikakken kwafi da aka gano/mL (± SE).
Bayan haka, mun bincika asalin ccfDNA a cikin mussels da aka tattara daga gadaje na mussel a tsibirin Kerguelen, rukunin tsibirai masu nisa da ke da iyakacin tasirin ɗan adam.Don wannan dalili, cccDNA daga mussel hemolymphs an ware kuma an tsarkake su ta hanyoyin da aka saba amfani da su don tsarkake cccDNA na ɗan adam [32, 33].Mun gano cewa matsakaicin adadin hemolymph ccfDNA a cikin mussels suna cikin ƙananan micrograms a kowace ML na kewayon haemolymph (duba Table S2, Ƙarin Bayani).Wannan kewayon adadin ya fi girma fiye da na mutane masu lafiya (ƙananan nanograms a kowace milliliter), amma a lokuta masu wuya, a cikin marasa lafiya na ciwon daji, matakin ccfDNA na iya kaiwa da yawa micrograms a kowace millilita [34, 35].Wani bincike na girman rabon hemolymph ccfDNA ya nuna cewa waɗannan gutsuttsura sun bambanta da girmansu, daga 1000 bp zuwa 1000 bp.har zuwa 5000 bp (Fig. 2).An sami irin wannan sakamakon ta amfani da Kit ɗin Binciken QIAamp na tushen silica, hanyar da aka saba amfani da ita a cikin ilimin kimiyyar shari'a don ware da sauri da tsarkake DNA daga ƙananan samfuran DNA, gami da ccfDNA [36].
Wakilin ccfDNA electrophoregram na mussel hemolymph.An cire shi tare da Kit ɗin Plasma NucleoSnap (saman) da Kit ɗin Binciken DNA na QIAamp.Makircin B Violin yana nuna rarraba adadin haemolymph ccfDNA (± SE) a cikin mussels.Layukan baki da ja suna wakiltar tsaka-tsaki da na farko da na uku, bi da bi.
Kusan 1% na ccfDNA a cikin mutane da primates suna da tushen waje [21, 37].Idan aka yi la'akari da tsarin zagaye-dabi-bude na bivalves, ruwan teku mai wadatar microbial, da girman rarraba mussel ccfDNA, mun yi hasashen cewa mussel hemolymph ccfDNA na iya ƙunsar tafki mai ɗimbin yawa na DNA microbial.Don gwada wannan hasashe, mun jera hemolymph ccfDNA daga samfuran Aulacomya atra da aka tattara daga tsibiran Kerguelen, wanda ya ba da fiye da karantawa miliyan 10, 97.6% waɗanda suka wuce ingantaccen sarrafawa.Sannan an rarraba karatun bisa ga tushen kai da waɗanda ba na kai ba ta amfani da bayanan BLASTN da NCBI bivalve (Fig. S1, Ƙarin Bayani).
A cikin mutane, duka biyun nukiliya da DNA na mitochondrial na iya fitowa cikin jini [38].Duk da haka, a cikin binciken da aka yi, ba zai yiwu a kwatanta dalla-dalla game da kwayar halittar DNA na mussels ba, ganin cewa ba a jera ko siffanta A. atra genome ba.Koyaya, mun sami damar gano wasu gutsuttsuran ccfDNA na asalin mu ta amfani da ɗakin karatu na bivalve (Fig. S2, Ƙarin Bayani).Mun kuma tabbatar da kasancewar guntuwar DNA na asalinmu ta hanyar haɓaka PCR na waɗannan kwayoyin halittar A. atra waɗanda aka jera (Fig. 3).Hakazalika, idan aka ba da cewa mitochondrial genome na A. atra yana samuwa a cikin bayanan jama'a, wanda zai iya samun shaida don kasancewar mitochondrial ccfDNA gutsuttsura a cikin hemolymph na A. atra.An tabbatar da kasancewar sassan DNA na mitochondrial ta hanyar haɓakawa na PCR (Fig. 3).
Dabbobin mitochondrial daban-daban sun kasance a cikin hemolymph na A. atra (dige ja - lambar hannun jari: SRX5705969) da M. platensis (dige shuɗi - lambar hannun jari: SRX5705968) haɓaka ta PCR.Hoton da aka daidaita daga Breton et al., 2011 B Ƙarawa na hemolymph supernatant daga A. atra Ajiye akan takarda FTA.Yi amfani da naushi mm 3 don ƙara kai tsaye zuwa bututun PCR mai ɗauke da haɗin PCR.
Idan aka ba da wadataccen abun ciki na ƙananan ƙwayoyin cuta a cikin ruwan teku, da farko mun mai da hankali kan halayen ƙwayoyin ƙwayoyin cuta na DNA a cikin hemolymph.Don yin wannan, muna amfani da dabaru daban-daban guda biyu.Dabarar farko ta yi amfani da Kraken2, wani shiri na tushen algorithm wanda zai iya gano jerin ƙananan ƙwayoyin cuta tare da daidaito mai kama da BLAST da sauran kayan aikin [28].Fiye da karatun 6719 an ƙaddara su zama asalin kwayoyin cuta, yayin da 124 da 64 sun fito ne daga archaea da ƙwayoyin cuta, bi da bi (Fig. 4).Mafi yawan ɓangarorin DNA na kwayan cuta sune Firmicutes (46%), Proteobacteria (27%), da Bacteroidates (17%) (Fig. 4a).Wannan rarraba ya yi daidai da binciken da ya gabata na marine blue mussel microbiome [39, 40].Gammaproteobacteria sune babban ajin Proteobacteria (44%), gami da Vibrionales da yawa (Fig. 4b).Hanyar ddPCR ta tabbatar da kasancewar Vibrio DNA gutsuttsura a cikin ccfDNA na A. atra hemolymph (Fig. 4c) [41].Don samun ƙarin bayani game da asalin kwayan cuta na ccfDNA, an ɗauki ƙarin hanya (Fig. S2, Ƙarin Bayani). A wannan yanayin, karanta abin da aka haɗu an haɗa su azaman karantawa-ƙarshen-ƙarshen kuma an rarraba su azaman na kai (bivalves) ko asalin kansa ta amfani da BLASTN da ƙimar e na 1e-3 da yanke tare da> 90% homology. A wannan yanayin, karanta abin da aka haɗu an haɗa su azaman karantawa-ƙarshen-ƙarshen kuma an rarraba su azaman na kai (bivalves) ko asalin kansa ta amfani da BLASTN da ƙimar e na 1e-3 da yanke tare da> 90% homology. Комментарии к видео ворчатые молюски) или чужие по происхождению с использованием BLASTN и значения e 1e-3 da отсечения A wannan yanayin, an tattara karatun da aka jera a matsayin karatun gama-gari kuma an rarraba su azaman ɗan ƙasa (bivalve) ko na asali ta amfani da BLASTN da ƙimar e na 1e-3 da yanke tare da> 90% homology.在这种情况下，重叠的读数组装为配对末端读数，并使用BLASTN 和1e-3的e 值和>90%为自身（双壳类）或非自身来源。在 用 情况 下 ， 重叠 读数 组装 为 配 末端 读数 ，源性 的 分类 自身 （双 壳类） 非 自身。 Комментарии к видео песни тые моллюски) или несобственные по происхождению A wannan yanayin, an tattara karatun da aka haɗa a matsayin karatun gama-gari kuma an rarraba su azaman nasu (bivalves) ko na asali ta amfani da e BLASTN da ƙimar 1e-3 da ƙimar homology> 90%.Tun da A. atra genome har yanzu ba a jera shi ba, mun yi amfani da dabarun taro na de novo na mai tarawa MEGAHIT na gaba Generation Sequencing (NGS).An gano jimlar 147,188 contigs a matsayin masu dogaro (bivalves) na asali.Daga nan sai aka fashe waɗancan abubuwan haɗin gwiwar tare da ƙimar e-1e-10 ta amfani da BLASTN da BLASTX.Wannan dabarar ta ba mu damar gano gutsuttsura 482 marasa bivalve da ke cikin A. atra ccfDNA.Fiye da rabin (57%) na waɗannan gutsuttssun DNA an samo su ne daga ƙwayoyin cuta, galibi daga gill symbionts, gami da sulfotrophic symbionts, kuma daga gill symbionts Solemya velum (Fig. 5).
Yawancin dangi a matakin nau'in.B Bambancin ƙananan ƙwayoyin cuta na manyan phyla guda biyu (Firmicutes da Proteobacteria).Wakilin haɓakawa na ddPCR C Vibrio spp.A. Gwargwadon kwayar halittar 16S rRNA (blue) a cikin haemolymphs guda uku.
An yi nazarin jimlar 482 da aka tattara.Bayanin gaba ɗaya na rarraba haraji na metagenomic contig annotations (prokaryotes da eukaryotes).B Cikakken rarraba gutsuwar DNA na kwayan cuta da BLASTN da BLASTX suka gano.
Binciken Kraken2 ya kuma nuna cewa mussel ccfDNA ya ƙunshi guntun DNA na archaeal, ciki har da gutsuttsuran DNA na Euryarchaeota (65%), Crenarchaeota (24%), da Thaurmarcheota (11%) (Fig. 6a).Kasancewar gutsuwar DNA da aka samo daga Euryarchaeota da Crenarchaeota, waɗanda aka samo a baya a cikin ƙananan ƙwayoyin cuta na mussels na California, bai kamata ya zo da mamaki ba [42].Kodayake Euryarchaeota ana danganta shi da matsananciyar yanayi, yanzu an gane cewa duka Euryarchaeota da Crenarcheota suna cikin prokaryotes na yau da kullun a cikin yanayin cryogenic na ruwa [43, 44].Kasancewar kwayoyin halittar methanogenic a cikin mussels ba abin mamaki bane, idan aka ba da rahotannin baya-bayan nan na kwararar methane mai yawa daga leaks na kasa a kan Kerguelen Plateau [45] da yiwuwar samar da methane na microbial da aka lura a bakin tekun tsibirin Kerguelen [46].
Daga nan hankalinmu ya koma ga karatu daga ƙwayoyin cuta na DNA.A iyakar saninmu, wannan shine bincike na farko da aka yi niyya na abubuwan da ke tattare da ƙwayoyin cuta na mussels.Kamar yadda aka sa ran, mun sami sassan DNA na bacteriophages (Caudovirales) (Fig. 6b).Duk da haka, mafi yawan kwayar cutar kwayar cutar kwayar cutar kwayar cutar kwayar cutar DNA ta fito ne daga phylum na nucleocytoviruses, wanda kuma aka sani da kwayar cutar cytoplasmic babba na DNA (NCLDV), wacce ke da mafi girman kwayar halitta ta kowace cuta.A cikin wannan phylum, yawancin jerin DNA suna cikin iyalai Mimimidoviridae (58%) da Poxviridae (21%), waɗanda rundunonin halitta sun haɗa da vertebrates da arthropods, yayin da ƙaramin rabo na waɗannan jerin DNA na cikin sanannun algae virological.Yana cutar da marine eukaryotic algae.Hakanan an samo jerin abubuwan ne daga kwayar cutar Pandora, babbar kwayar cutar da ke da girman kwayar halitta mafi girma na kowane nau'in kwayar cuta da aka sani.Abin sha'awa, kewayon rundunonin da aka sani suna kamuwa da ƙwayar cuta, kamar yadda tsarin hemolymph ccfDNA ya ƙaddara, ya yi girma sosai (Hoto S3, Ƙarin Bayani).Ya haɗa da ƙwayoyin cuta masu cutar da kwari irin su Baculoviridae da Iridoviridae, da ƙwayoyin cuta masu cutar da amoeba, algae da vertebrates.Mun kuma sami jerin abubuwan da suka dace da Pithovirus sibericum genome.Pitoviruses (wanda kuma aka sani da "kwayoyin cutar zombie") an fara keɓe su daga permafrost mai shekaru 30,000 a Siberiya [47].Don haka, sakamakonmu ya yi daidai da rahotannin da suka gabata suna nuna cewa ba dukkanin nau'ikan ƙwayoyin cuta na zamani ba ne ke bacewa [48] kuma waɗannan ƙwayoyin cuta na iya kasancewa a cikin yanayin yanayin teku mai nisa.
A ƙarshe, mun gwada don ganin ko za mu iya samun gutsuwar DNA daga wasu dabbobi masu yawa.BLASTN da BLASTX sun gano jimlar 482 na ƙasashen waje tare da nt, nr da ɗakunan karatu na RefSeq (genomic da furotin).Sakamakonmu ya nuna cewa a cikin ɓangarorin ƙasashen waje na ccfDNA na dabbobi masu yawa DNA na ƙasusuwan ƙashi sun fi rinjaye (Fig. 5).An kuma gano gutsure DNA daga kwari da sauran nau'ikan.Ba a gano wani yanki mai girma na gutsuttsarin DNA ba, mai yiyuwa ne saboda rashin wakilcin adadi mai yawa na nau'in ruwa a cikin bayanan kwayoyin halitta idan aka kwatanta da nau'in terrestrial [49].
A cikin takarda na yanzu, mun yi amfani da ra'ayin LB ga mussels, suna jayayya cewa tsarin harbi na hemolymph ccfDNA na iya ba da haske game da abubuwan da ke tattare da yanayin yanayin teku.Musamman ma, mun gano cewa 1) hemolymph mussel ya ƙunshi ƙananan ƙananan ƙwayoyin cuta (matakan microgram) na ƙananan ƙananan (~ 1-5 kb) rarraba DNA gutsuttsura;2) waɗannan guntuwar DNA suna da masu zaman kansu da marasa zaman kansu 3) Daga cikin tushen ƙasashen waje na waɗannan gutsuttsaran DNA, mun sami DNA na ƙwayoyin cuta, archaeal da viral DNA, da DNA na sauran dabbobi masu yawa;4) Tarin waɗannan ɓangarorin ccfDNA na ƙasashen waje a cikin hemolymph yana faruwa da sauri kuma yana ba da gudummawa ga ayyukan tacewa na ciki na mussels.A ƙarshe, bincikenmu ya nuna cewa manufar LB, wanda ya zuwa yanzu an yi amfani da shi musamman a fannin nazarin halittu, ya ƙunshi tushen ilimi mai wadata amma ba a gano shi ba wanda za a iya amfani da shi don ƙarin fahimtar hulɗar da ke tsakanin nau'in sentinel da muhallinsu.
Baya ga primates, an ba da rahoton keɓewar ccfDNA a cikin dabbobi masu shayarwa, gami da beraye, karnuka, kuliyoyi, da dawakai [50, 51, 52].Koyaya, a cikin iliminmu, bincikenmu shine farkon wanda ya ba da rahoton ganowa da jerin abubuwan ccfDNA a cikin nau'ikan ruwa tare da tsarin buɗe ido.Wannan siffa ta jiki da ikon tacewa na mussels na iya, aƙalla a wani ɓangare, ya bayyana nau'ikan girman nau'ikan nau'ikan rarrabawar DNA idan aka kwatanta da sauran nau'ikan.A cikin mutane, yawancin gutsuttsuran DNA da ke yawo a cikin jini ƙananan guntu ne masu girma daga 150 zuwa 200 bp.tare da iyakar iyakar 167 bp [34, 53].Ƙananan yanki amma mahimmanci na sassan DNA suna tsakanin 300 da 500 bp a girman, kuma kusan 5% sun fi 900 bp tsawo.[54]Dalilin wannan girman rarraba shine cewa babban tushen ccfDNA a cikin plasma yana faruwa ne sakamakon mutuwar kwayar halitta, ko dai saboda mutuwar tantanin halitta ko kuma saboda necrosis na ƙwayoyin hematopoietic da ke yawo a cikin mutane masu lafiya ko kuma saboda apoptosis na ƙwayoyin tumo a cikin marasa lafiya na ciwon daji (wanda aka sani da circulating tumor DNA).ctDNA).Girman rabon hemolymph ccfDNA da muka samu a cikin mussels ya bambanta daga 1000 zuwa 5000 bp, yana nuna cewa mussel ccfDNA yana da asali daban.Wannan hasashe ne na ma'ana, tunda mussels suna da tsarin jijiyoyin jini na buɗaɗɗen buɗe ido kuma suna rayuwa a cikin mahalli na ruwa na ruwa mai ɗauke da adadi mai yawa na DNA na ƙwayoyin cuta.A gaskiya ma, gwaje-gwajenmu na dakin gwaje-gwaje ta amfani da DNA na waje sun nuna cewa mussels suna tara gutsuttsura DNA a cikin ruwan teku, aƙalla bayan 'yan sa'o'i kadan sun lalace bayan ɗaukar salula da / ko saki da / ko adana su a cikin kungiyoyi daban-daban.Ganin ƙarancin ƙwayoyin sel (duka prokaryotic da eukaryotic), yin amfani da ɓangarori na intravalvular zai rage adadin ccfDNA daga tushen kai da kuma daga ƙasashen waje.Idan aka yi la'akari da mahimmancin rigakafi na bivalve da kuma yawan adadin phagocytes masu yawo, mun ƙara yin hasashen cewa hatta ccfDNA na ƙasashen waje an wadatar da su a cikin yaɗa phagocytes waɗanda ke tara DNA na ƙasashen waje yayin cin ƙwayoyin ƙwayoyin cuta da/ko tarkace ta salula.A hade, sakamakonmu ya nuna cewa bivalve hemolymph ccfDNA wani ma'auni ne na musamman na bayanan kwayoyin halitta kuma yana ƙarfafa matsayinsu na nau'in sentinel.
Bayananmu sun nuna cewa jeri da nazarin gutsuttsuran hemolymph ccfDNA da aka samu daga ƙwayoyin cuta na iya ba da mahimman bayanai game da flora na ƙwayoyin cuta da ƙwayoyin cuta da ke cikin yanayin yanayin ruwa da ke kewaye.Dabarun jeri na harbi sun bayyana jerin jeri na ƙwayoyin cuta A. atra gill waɗanda da an rasa su idan an yi amfani da hanyoyin ganowa na 16S rRNA na al'ada, saboda wani ɓangare na nuna son rai na ɗakin karatu.A gaskiya ma, amfani da bayanan LB da aka tattara daga M. platensis a cikin nau'in mussel guda a Kerguelen ya nuna cewa abubuwan da ke tattare da gill da ke hade da kwayar cutar kwayar cutar ta kasance iri ɗaya ga nau'in mussel (Fig. S4, Ƙarin Bayani).Wannan kamanceceniya na mussels daban-daban guda biyu na iya yin nuni da abun da ke tattare da al'ummomin kwayoyin cuta a cikin sanyi, sulfurous, da tudun wuta na Kerguelen [55, 56, 57, 58].An bayyana mafi girman matakan ƙwayoyin cuta masu rage sulfur da kyau lokacin da ake girbin kayan lambu daga yankunan bakin teku da suka lalace [59], kamar gabar tekun Port-au-Faransa.Wata yuwuwar kuma ita ce commensal flora mussel na iya shafar watsawa a kwance [60, 61].Ana buƙatar ƙarin bincike don tantance alaƙa tsakanin yanayin ruwa, saman bene na teku, da abun da ke tattare da ƙwayoyin cuta na siminti a cikin mussels.Wadannan karatun suna gudana a halin yanzu.
Tsawon da tattarawar hemolymph ccfDNA, sauƙin tsarkakewa, da inganci mai kyau don ba da izinin jerin harbe-harbe cikin sauri wasu fa'idodi da yawa na amfani da mussel ccfDNA don tantance bambancin halittu a cikin yanayin tekun teku.Wannan tsarin yana da tasiri musamman don siffanta al'ummomin hoto (viromes) a cikin yanayin da aka ba da shi [62, 63].Ba kamar ƙwayoyin cuta, archaea, da eukaryotes ba, ƙwayoyin cuta na hoto ko bidiyo mai zagaya yanar gizo da sauri ba su ƙunshe da kwayoyin halittar da aka kiyaye su ba kamar jerin 16S.Sakamakonmu ya nuna cewa ana iya amfani da biopsies na ruwa daga nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan mussels kamar mussels don gano adadi mai yawa na gutsuttsuran ƙwayoyin cuta na ccfDNA da aka sani suna cutar da rundunonin da ke mamaye yanayin yanayin tekun teku.Wannan ya haɗa da ƙwayoyin cuta da aka sani suna cutar da protozoa, arthropods, kwari, tsirrai, da ƙwayoyin cuta na kwayan cuta (misali, bacteriophages).An samo irin wannan rarraba lokacin da muka bincika hemolymph ccfDNA virome na blue mussels (M. platensis) da aka tattara a cikin mussel Layer a Kerguelen (Table S2, Ƙarin Bayani).Tsarin Shotgun na ccfDNA hakika sabuwar hanya ce da ke samun ci gaba a cikin binciken kwayar halittar mutane ko wasu nau'ikan [21, 37, 64].Wannan hanya tana da amfani musamman don nazarin ƙwayoyin cuta na DNA guda biyu, tun da babu wani nau'in kwayar halitta guda ɗaya da aka adana a cikin dukkanin ƙwayoyin cuta na DNA masu ɗaure biyu, wakiltar mafi yawan nau'ikan ƙwayoyin cuta da yawa a cikin Baltimore [65].Ko da yake yawancin waɗannan ƙwayoyin cuta ba su da alaƙa kuma suna iya haɗawa da ƙwayoyin cuta daga wani ɓangaren da ba a sani ba gaba ɗaya na duniyar hoto [66], mun gano cewa viromes da jeri na mussels A. atra da M. platensis sun faɗi tsakanin nau'in biyu.haka (duba adadi S3, ƙarin bayani).Wannan kamanceceniya ba abin mamaki bane, domin yana iya nuna rashin zaɓin zaɓi a cikin ɗaukar DNA da ke cikin muhalli.Ana buƙatar karatun gaba ta amfani da RNA mai tsafta a halin yanzu don siffanta ƙwayar RNA.
A cikin bincikenmu, mun yi amfani da wani bututu mai tsauri wanda aka daidaita daga aikin Kowarski da abokan aiki [37], waɗanda suka yi amfani da share matakai guda biyu na abubuwan karantawa da ƙira kafin da kuma bayan taron ccfDNA na asali, wanda ya haifar da adadi mai yawa na karantawa ba tare da taswira ba.Saboda haka, ba za mu iya yin watsi da cewa wasu daga cikin waɗannan karatun da ba a taswira ba suna iya samun nasu asali, da farko saboda ba mu da wani nau'in kwayar halitta na wannan nau'in mussel.Mun kuma yi amfani da wannan bututun saboda mun damu da chimeras tsakanin karatun kai da wanda ba kai ba da kuma tsawon karatun da Illumina MiSeq PE75 ta samar.Wani dalili na yawancin karatun da ba a tantance shi ba shine yawancin ƙwayoyin cuta na ruwa, musamman a yankuna masu nisa kamar Kerguelen, ba a bayyana su ba.Mun yi amfani da Illumina MiSeq PE75, muna ɗaukar tsayin guntun ccfDNA kama da ccfDNA na ɗan adam.Don karatu na gaba, idan aka ba da sakamakonmu da ke nuna cewa hemolymph ccfDNA yana da tsayin karatu fiye da mutane da/ko dabbobi masu shayarwa, muna ba da shawarar yin amfani da tsarin da ya fi dacewa da guntun ccfDNA mai tsayi.Wannan aikin zai sa ya fi sauƙi don gano ƙarin alamun bincike mai zurfi.Samun cikakken jerin kwayoyin halittar A. atra na nukiliya wanda babu shi a halin yanzu zai taimaka matuƙar sauƙaƙe wariyar ccfDNA daga tushen kai da waɗanda ba na kai ba.Ganin cewa bincikenmu ya mayar da hankali kan yuwuwar yin amfani da manufar biopsy na ruwa ga mussels, muna fatan cewa yayin da ake amfani da wannan ra'ayi a cikin bincike na gaba, za a samar da sabbin kayan aiki da bututun mai don haɓaka yuwuwar wannan hanyar don yin nazarin bambancin ƙwayoyin cuta na mussels.yanayin yanayin ruwa.
A matsayin biomarker na asibiti ba mai haɗari ba, haɓakar matakan plasma na ɗan adam na ccfDNA suna da alaƙa da cututtuka daban-daban, lalacewar nama, da yanayin damuwa [67,68,69].Wannan haɓaka yana da alaƙa da sakin sassan DNA na asalinsa bayan lalacewar nama.Mun magance wannan batu ta amfani da matsananciyar zafi, inda aka ɗan ɗanɗana ƙwanƙwasa ga zafin jiki na 30 ° C.Mun yi wannan bincike akan nau'ikan mussels guda uku a cikin gwaje-gwaje masu zaman kansu guda uku.Koyaya, ba mu sami wani canji a matakan ccfDNA ba bayan matsanancin matsanancin zafi (duba Hoto S5, ƙarin bayani).Wannan binciken na iya yin bayani, aƙalla a wani ɓangare, gaskiyar cewa mussels suna da tsarin jini na buɗe ido kuma suna tara adadi mai yawa na DNA na ƙasashen waje saboda yawan aikin tacewa.A gefe guda, mussels, kamar yawancin invertebrates, na iya zama mafi juriya ga lalacewar nama da ke haifar da damuwa, ta haka yana iyakance sakin ccfDNA a cikin hemolymph [70, 71].
Ya zuwa yau, nazarin DNA na bambancin halittu a cikin halittun ruwa ya fi mayar da hankali kan DNA na muhalli (eDNA) metabarcoding.Koyaya, wannan hanyar yawanci tana iyakance ne a cikin nazarin halittun halittu lokacin da ake amfani da firam.Yin amfani da jeri na harbin bindiga yana kewaye da iyakokin PCR da zaɓin son zuciya na saitin firamare.Don haka, a wata ma'ana, hanyarmu ta fi kusa da hanyar da aka yi amfani da ita kwanan nan ta hanyar eDNA Shotgun mai girma, wacce ke da ikon jera DNA rarrabke kai tsaye da bincika kusan dukkanin halittu [72, 73].Koyaya, akwai wasu batutuwa masu mahimmanci waɗanda suka bambanta LB daga daidaitattun hanyoyin eDNA.Tabbas, babban bambanci tsakanin eDNA da LB shine amfani da runduna masu tacewa na halitta.An ba da rahoton yin amfani da nau'ikan ruwa kamar soso da bivalves (Dresseina spp.) azaman tacewa na halitta don nazarin eDNA [74, 75].Duk da haka, binciken Dreissena ya yi amfani da biopsies na nama wanda aka fitar da DNA.Binciken ccfDNA daga LB baya buƙatar biopsy nama, na musamman kuma wani lokacin tsada kayan aiki da dabaru masu alaƙa da eDNA ko biopsy nama.A gaskiya ma, kwanan nan mun ba da rahoton cewa ccfDNA daga LB za a iya adanawa da kuma nazarin su tare da goyon bayan FTA ba tare da kiyaye sarkar sanyi ba, wanda shine babban kalubale ga bincike a wurare masu nisa [76].Cire ccfDNA daga biopsies na ruwa shima mai sauƙi ne kuma yana samar da DNA mai inganci don jerin bindigogi da bincike na PCR.Wannan babbar fa'ida ce da aka ba da wasu iyakokin fasaha da ke da alaƙa da nazarin eDNA [77].Sauƙi da ƙarancin farashi na hanyar yin samfur kuma sun dace musamman don shirye-shiryen sa ido na dogon lokaci.Baya ga babban ƙarfin tacewa, wani sanannen fasalin bivalves shine sinadari na mucopolysaccharide na gabobin su, wanda ke haɓaka ɗaukar ƙwayoyin cuta [78, 79].Wannan ya sa bivalves ya zama madaidaicin tacewa na halitta don kwatanta bambancin halittu da tasirin canjin yanayi a cikin yanayin yanayin ruwa da aka ba da.Ko da yake ana iya ganin kasancewar guntuwar DNA da aka samu mai masaukin baki a matsayin iyakancewar hanyar idan aka kwatanta da eDNA, farashin da ke da alaƙa da samun irin wannan ɗan asalin ccfDNA idan aka kwatanta da eDNA abu ne mai sauƙin fahimta a lokaci guda don ɗimbin bayanan da ke akwai don nazarin lafiya.biya diyya mai masaukin baki.Wannan ya haɗa da kasancewar jerin ƙwayoyin cuta da aka haɗa a cikin kwayoyin halittar mai watsa shiri.Wannan yana da mahimmanci musamman ga mussels, idan aka ba da kasancewar ƙwayoyin cutar leukemia retroviruses a kwance a cikin bivalves [80, 81].Wani fa'idar LB akan eDNA shine cewa yana amfani da aikin phagocytic na zagayawan ƙwayoyin jini a cikin hemolymph, wanda ke mamaye ƙwayoyin cuta (da kwayoyin halittarsu).Phagocytosis shine babban aikin ƙwayoyin jini a cikin bivalves [82].A ƙarshe, hanyar tana amfani da babban ƙarfin tacewa na mussels (matsakaicin 1.5 l / h na ruwan teku) da kuma wurare dabam dabam na kwana biyu, wanda ke haɓaka haɗuwa da yadudduka daban-daban na ruwan teku, yana ba da damar kama eDNA heterologous.[83, 84].Don haka, binciken mussel ccfDNA hanya ce mai ban sha'awa da aka ba da tasirin sinadirai, tattalin arziki, da muhalli na mussels.Hakazalika da nazarin LB da aka tattara daga mutane, wannan hanya kuma ta buɗe yiwuwar auna kwayoyin halitta da sauye-sauye na epigenetic a cikin DNA mai watsa shiri don amsawa ga abubuwan da ba su da kyau.Misali, ana iya yin hasashen fasahohin jeri na ƙarni na uku don yin nazarin methylation mai faɗi a cikin ccfDNA na asali ta amfani da jerin nanopore.Ya kamata a sauƙaƙe wannan tsari ta hanyar gaskiyar cewa tsayin ɓangarorin mussel ccfDNA ya dace daidai da tsarin dandali mai tsayi da aka karanta wanda ke ba da damar nazarin DNA methylation na genome-fadi daga jerin guda ɗaya ba tare da buƙatar sauye-sauyen sinadarai ba.85,86] Wannan abu ne mai ban sha'awa mai yiwuwa, kamar yadda aka nuna cewa DNA methylation alamu da kuma ci gaba da mayar da martani ga yawancin tsararru na muhalli.Sabili da haka, yana iya ba da haske mai mahimmanci game da hanyoyin da ke haifar da amsawa bayan bayyanar da canjin yanayi ko gurɓatawa [87].Koyaya, amfani da LB ba tare da iyakancewa ba ne.Ba lallai ba ne a faɗi, wannan yana buƙatar kasancewar nau'ikan alamomi a cikin yanayin yanayin.Kamar yadda aka ambata a sama, yin amfani da LB don tantance bambancin halittu na yanayin da aka ba da shi kuma yana buƙatar bututun bioinformatics mai tsauri wanda yayi la'akari da kasancewar gutsutsayen DNA daga tushen.Wata babbar matsala ita ce samuwar kwayoyin halittun da ake amfani da su a cikin ruwa.Ana fatan yunƙuri irin su na Marine Mammal Genomes Project da aikin Fish10k da aka kafa kwanan nan [88] za su sauƙaƙe irin wannan bincike a nan gaba.Aiwatar da ra'ayi na LB zuwa ga kwayoyin ciyar da matatun ruwa kuma ya dace da sabbin ci gaba a cikin fasahar kere-kere, wanda ya sa ya dace sosai don haɓaka masana'antar biomarkers da yawa don samar da mahimman bayanai game da lafiyar wuraren zama 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