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Binciken ruwa (LB) ra'ayi ne da ke samun karbuwa cikin sauri a fannin ilmin halitta. Manufar ta dogara ne akan gano gutsuttsuran DNA na waje (ccfDNA), waɗanda galibi ake saki a matsayin ƙananan gutsuttsuran bayan mutuwar ƙwayoyin halitta a cikin kyallen takarda daban-daban. Ƙaramin kaso na waɗannan gutsuttsuran sun samo asali ne daga kyallen takarda ko halittu na waje (na ƙasashen waje). A cikin aikin da muke yi a yanzu, mun yi amfani da wannan ra'ayi ga mussels, wani nau'in tsaro da aka sani da ƙarfin tace ruwan teku. Muna amfani da ikon mussels don yin aiki azaman matatun halitta don kama gutsuttsuran DNA na muhalli daga tushe daban-daban don samar da bayanai game da bambancin halittu na yanayin halittu na bakin teku. Sakamakonmu ya nuna cewa mussel hemolymph ya ƙunshi gutsuttsuran DNA waɗanda suka bambanta sosai a girma, daga 1 zuwa 5 kb. Jerin bindigogin harbi ya nuna cewa adadi mai yawa na gutsuttsuran DNA asalin ƙwayoyin cuta ne na ƙasashen waje. Daga cikinsu, mun sami gutsuttsuran DNA daga ƙwayoyin cuta, archaea, da ƙwayoyin cuta, gami da ƙwayoyin cuta da aka sani suna kamuwa da nau'ikan masu masaukin baki da aka saba samu a cikin yanayin halittu na bakin teku. A ƙarshe, bincikenmu ya nuna cewa manufar LB da aka yi amfani da ita ga mussels tana wakiltar tushen ilimi mai yawa amma ba a bincika ba tukuna game da bambancin ƙwayoyin cuta a cikin yanayin halittu na bakin teku.
Tasirin sauyin yanayi (CC) akan bambancin halittu na halittun ruwa wani fanni ne da ke ci gaba da bunƙasa cikin sauri na bincike. Dumamar yanayi ba wai kawai yana haifar da muhimman matsalolin ilimin halittar jiki ba, har ma yana tura iyakokin juyin halitta na kwanciyar hankali na yanayin zafi na halittun ruwa, yana shafar mazaunin wasu nau'ikan halittu, yana sa su nemi ƙarin yanayi mai kyau [1, 2]. Baya ga shafar bambancin halittu na metazoans, CC yana kawo cikas ga daidaiton hulɗar mai masaukin baki da ƙwayoyin cuta. Wannan dysbacteriosis na ƙwayoyin cuta yana haifar da babbar barazana ga halittun ruwa yayin da yake sa halittun ruwa su fi saurin kamuwa da cututtukan da ke yaɗuwa [3, 4]. Ana kyautata zaton cewa SS yana taka muhimmiyar rawa a mutuwar mutane da yawa, wanda matsala ce mai tsanani ga kula da halittun ruwa na duniya [5, 6]. Wannan batu ne mai mahimmanci idan aka yi la'akari da tasirin tattalin arziki, muhalli da abinci mai gina jiki na nau'ikan ruwa da yawa. Wannan gaskiya ne musamman ga bivalves da ke zaune a yankunan polar, inda tasirin CK ya fi tsanani da sauri [6, 7]. A gaskiya ma, ana amfani da bivalves kamar Mytilus spp. sosai don sa ido kan tasirin CC akan halittun ruwa. Ba abin mamaki ba ne, an ƙirƙiro adadi mai yawa na biomarkers don sa ido kan lafiyarsu, galibi suna amfani da hanyar matakai biyu da ta ƙunshi biomarkers masu aiki bisa ga ayyukan enzymatic ko ayyukan ƙwayoyin halitta kamar yuwuwar ƙwayoyin halitta da aikin phagocytic [8]. Waɗannan hanyoyin sun haɗa da auna yawan takamaiman alamun matsin lamba waɗanda ke taruwa a cikin kyallen takarda masu laushi bayan shan ruwa mai yawa na teku. Duk da haka, babban ƙarfin tacewa da tsarin zagayawa na bivalves mai buɗewa suna ba da dama don haɓaka sabbin biomarkers na hemolymph ta amfani da manufar biopsy na ruwa (LB), hanya mai sauƙi kuma mai sauƙin mamayewa don kula da marasa lafiya. samfuran jini [9, 10]. Kodayake ana iya samun nau'ikan ƙwayoyin da ke zagayawa da yawa a cikin LB na ɗan adam, wannan ra'ayi ya dogara ne akan nazarin jerin DNA na gutsuttsuran DNA na ƙwayoyin halitta (ccfDNA) da ke zagayawa a cikin plasma. A zahiri, an san kasancewar DNA mai zagayawa a cikin plasma na ɗan adam tun tsakiyar ƙarni na 20 [11], amma a cikin 'yan shekarun nan ne kawai zuwan hanyoyin jerin abubuwa masu ƙarfi ya haifar da ganewar asibiti bisa ga ccfDNA. Kasancewar waɗannan gutsuttsuran DNA da ke yawo a jiki ya faru ne sakamakon sakin DNA na genomic (nukiliya da mitochondria) bayan mutuwar ƙwayoyin halitta. A cikin mutane masu lafiya, yawan ccfDNA yawanci yana da ƙasa (<10 ng/mL) amma ana iya ƙara shi sau 5-10 a cikin marasa lafiya da ke fama da cututtuka daban-daban ko waɗanda ke fuskantar damuwa, wanda ke haifar da lalacewar nama. A cikin mutane masu lafiya, yawan ccfDNA yawanci yana da ƙasa (<10 ng/mL) amma ana iya ƙara shi sau 5-10 a cikin marasa lafiya da ke fama da cututtuka daban-daban ko waɗanda ke 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 различной патологией или подвергающихся стрессу, приводящему к повреждению тканей. A cikin mutane masu lafiya, yawan sinadarin cccDNA yawanci yana da ƙasa (<10 ng/mL), amma yana iya ƙaruwa da sau 5-10 a cikin marasa lafiya da ke da cututtuka daban-daban ko kuma waɗanda ke 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）。倍，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 种增加 5-10 倍 从而 组织。 损伤 损伤Концентрации ccfDNA обычно низкие (<10 нг/мл) различными патологиями или стрессом, что приводит к повреждению тканей. Yawan ccfDNA yawanci yana da ƙasa (<10 ng/ml) a cikin mutane masu lafiya, amma ana iya ƙara shi sau 5-10 a cikin marasa lafiya da ke da cututtuka daban-daban ko damuwa, wanda ke haifar da lalacewar nama.Girman gutsuttsuran ccfDNA ya bambanta sosai, amma yawanci yana tsakanin 150 zuwa 200 bp. [12]. Ana iya amfani da nazarin ccfDNA da aka samo asali daga kansa, watau, ccfDNA daga ƙwayoyin halitta na yau da kullun ko waɗanda aka canza, don gano canje-canjen kwayoyin halitta da epigenetic da ke cikin kwayar halittar nukiliya da/ko mitochondrial, don haka yana taimaka wa likitoci su zaɓi takamaiman hanyoyin magance ƙwayoyin cuta [13]. Duk da haka, ana iya samun ccfDNA daga kafofin waje kamar ccfDNA daga ƙwayoyin tayi yayin daukar ciki ko daga gabobin da aka dasa [14,15,16,17]. ccfDNA kuma muhimmin tushen bayanai ne don gano kasancewar nucleic acid na wani wakili mai kamuwa da cuta (na ƙasashen waje), wanda ke ba da damar gano cututtukan da ba a iya kamuwa da su ba ta hanyar al'adun jini, yana guje wa biopsy mai mamaye nama mai kamuwa da cuta [18]. Binciken da aka yi kwanan nan ya nuna cewa jinin ɗan adam yana ɗauke da wadataccen tushen bayanai wanda za a iya amfani da shi don gano ƙwayoyin cuta da ƙwayoyin cuta, kuma kusan 1% na ccfDNA da aka samu a cikin jinin ɗan adam asalinsa ne na ƙasashen waje [19]. Waɗannan nazarin sun nuna cewa ana iya tantance bambancin halittu na ƙwayoyin cuta masu yawo a cikin wani halitta ta amfani da nazarin ccfDNA. Duk da haka, har zuwa kwanan nan, wannan ra'ayi ana amfani da shi ne kawai a cikin mutane, kuma, a cikin ƙaramin ɓangare, a cikin sauran halittu masu ƙashi [20, 21].
A cikin wannan takarda, mun yi amfani da ƙarfin LB don yin nazarin ccfDNA na Aulacomya atra, wani nau'in kudanci da aka fi samu a Tsibirin Kerguelen mai zurfi, wani rukuni na tsibirai a saman babban tudu wanda ya samo asali shekaru miliyan 35 da suka gabata. fashewar aman wuta. Ta amfani da tsarin gwaji na in vitro, mun gano cewa gutsuttsuran DNA a cikin ruwan teku suna ɗaukar mussels cikin sauri kuma suna shiga cikin sashin hemolymph. Jerin bindigogi ya nuna cewa mussel hemolymph ccfDNA yana ɗauke da gutsuttsuran DNA na asalinsa da wanda ba na kansa ba, gami da ƙwayoyin cuta masu alaƙa da ƙwayoyin halittar ruwa masu sanyi da gutsuttsuran DNA daga halittun halittu masu kama da yanayin yanayin bakin teku na aman wuta. Hemolymph ccfDNA kuma yana ɗauke da jerin ƙwayoyin cuta da aka samo daga ƙwayoyin cuta tare da kewayon masu masaukin baki daban-daban. Mun kuma sami gutsuttsuran DNA daga dabbobi masu ƙwayoyin halitta da yawa kamar kifi mai ƙashi, anemones na teku, algae da kwari. A ƙarshe, bincikenmu ya nuna cewa ana iya amfani da ra'ayin LB cikin nasara ga invertebrates na ruwa don samar da tarin kwayoyin halitta masu yawa a cikin yanayin halittu na ruwa.
An tattara manya (tsawon mm 55-70) Mytilus platensis (M. platensis) da Aulacomya atra (A. atra) daga bakin teku mai duwatsu na Port-au-France (049°21.235 S, 070°13.490 E.). Tsibirin Kerguelen a watan Disamba na 2018. An samo wasu manyan mussels masu launin shuɗi (Mytilus spp.) daga mai samar da kayayyaki na kasuwanci (PEI Mussel King Inc., Prince Edward Island, Kanada) kuma an sanya su a cikin tanki mai iska mai zafi (4°C) wanda ke ɗauke da lita 10-20 na ruwan gishiri na wucin gadi 32‰. (gishirin teku na wucin gadi Reef Crystal, Instant Ocean, Virginia, Amurka). Ga kowane gwaji, an auna tsawon da nauyin harsashin mutum ɗaya.
Ana samun yarjejeniyar buɗewa kyauta don wannan shirin akan layi (https://doi.org/10.17504/protocols.io.81wgb6z9olpk/v1). A takaice, an tattara hemolymph na LB daga tsokoki na masu satar bayanai kamar yadda aka bayyana [22]. An fayyace hemolymph ɗin ta hanyar centrifugation a 1200 ×g na tsawon mintuna 3, an daskare ruwan da ke cikinsa (-20°C) har sai an yi amfani da shi. Don warewa da tsarkake cfDNA, an narke samfuran (1.5-2.0 ml) kuma an sarrafa su ta amfani da kayan aikin NucleoSnap cfDNA (Macherey-Nagel, Bethlehen, PA) bisa ga umarnin masana'anta. An adana ccfDNA a -80°C har sai an ci gaba da bincike. A wasu gwaje-gwaje, an ware ccfDNA kuma an tsarkake ta ta amfani da Kayan Binciken DNA na QIAamp (QIAGEN, Toronto, Ontario, Kanada). An auna DNA mai tsabta ta amfani da gwajin PicoGreen na yau da kullun. An yi nazarin rarrabawar gutsuttsuran ccfDNA da aka keɓe ta hanyar amfani da na'urar auna ƙwayoyin cuta ta Agilent 2100 (Agilent Technologies Inc., Santa Clara, CA) ta amfani da Kayan Aikin DNA Mai Jin Daɗi. An yi gwajin ta amfani da µl na samfurin ccfDNA bisa ga umarnin masana'anta.
Don tsara guntun ccfDNA na hemolymph, Génome Québec (Montreal, Quebec, Kanada) sun shirya ɗakunan karatu na bindiga ta amfani da kayan haɗin jini na Illumina DNA na kayan aikin Illumina MiSeq PE75. An yi amfani da adaftar daidaitacce (BioO). Ana samun fayilolin bayanai na asali daga NCBI Sequence Read Archive (SRR8924808 da SRR8924809). An tantance ingancin karatu na asali ta amfani da FastQC [23]. An yi amfani da Trimmomatic [24] don yanke adaftar da kuma karanta marasa inganci. An haɗa karatun bindiga tare da ƙarshen da aka haɗa FLASH zuwa cikin dogon karatu ɗaya tare da mafi ƙarancin haɗuwa na 20 bp don guje wa rashin daidaito [25]. An yi wa karatun da aka haɗa alama da BLASTN ta amfani da bayanan NCBI Taxonomy mai bivalve (ƙimar e < 1e−3 da 90% homology), kuma an yi ɓoye jerin abubuwan da ba su da rikitarwa ta amfani da DUST [26]. An yi wa karatun da aka haɗa alama da BLASTN ta amfani da bayanan NCBI Taxonomy mai bivalve (ƙimar e < 1e−3 da 90% homology), kuma an yi ɓoye jerin abubuwan da ba su da rikitarwa ta amfani da DUST [26]. Объединенные чтения были аннотированы с помощью BLASTN смотреть NCBI (значение e <1e-3 da 90% гомологии), а маскирование последовательностей. [26]. An yi wa karatun da aka tattara sunaye lakabi da BLASTN ta amfani da bayanan NCBI bivalve taxonomy (ƙimar e < 1e-3 da 90% homology), kuma an yi ɓoye jerin abubuwa masu ƙarancin rikitarwa ta amfani da DUST [26].使用双壳类NCBI 分类数据库（e 值< 1e-3 和90% 同源性）用BLASTN 注释合并的读据库进行低复杂度序列的掩蔽。使用 双 壳类 ncbi 分类 （（（<1e-3 和 90% 同源）进行复杂度 序列 的。。。 掩蔽 掩蔽掩蔽 掩蔽 掩蔽Объединенные чтения были аннотированы с помощью BLASTN с использованием моллюсков NCBI (значение e <1e-3 и 90% гомологии), а маскирование последовательностей. использованием KURA [26]. An yi wa karatun da aka tattara sunaye lakabi da BLASTN ta amfani da bayanan NCBI bivalve taxonomic (ƙimar e <1e-3 da 90% homology), kuma an yi ɓoye jerin abubuwa masu ƙarancin rikitarwa ta amfani da DUST [26].An raba karatun zuwa ƙungiyoyi biyu: masu alaƙa da jerin bivalve (a nan ana kiran su da karatun kai) da kuma waɗanda ba su da alaƙa (karanta kai). An haɗa ƙungiyoyi biyu daban-daban ta amfani da MEGAHIT don samar da contigs [27]. A halin yanzu, an rarraba rarrabawar ƙididdigar ƙwayoyin cuta na baƙi ta amfani da Kraken2 [28] kuma an wakilta ta hanyar zane-zane ta hanyar jadawalin kek na Krona akan Galaxy [29, 30]. An ƙaddara cewa mafi kyawun kmers shine kmers-59 daga gwaje-gwajen farko da muka yi. An gano cututtukan da ke da alaƙa da kai ta hanyar daidaitawa da BLASTN (bayanan bivalve NCBI, e value < 1e−10 da 60% homology) don bayanin ƙarshe. An gano cututtukan da ke da alaƙa da kai ta hanyar daidaitawa da BLASTN (bayanan bivalve NCBI, e value < 1e−10 da 60% homology) don bayanin ƙarshe. Затем собственные контиги были идентифицированы путем сопоставления с BLASTN значение e <1e-10 и гомология 60%) для окончательной аннотации. An gano cututtukan da ke da alaƙa da kansu ta hanyar daidaitawa da BLASTN (bayanan bivalve na NCBI, ƙimar e <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%同源性）对齐来识别自身重叠群以进行最终注释。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 - для двустворчатых моллюсков, значение e <1e-10 и гомология 60%). An gano cututtukan da ke da alaƙa da kansu don yin bayani na ƙarshe ta hanyar daidaitawa da BLASTN (bayanan bivalve na NCBI, ƙimar e <1e-10 da 60% homology). A lokaci guda, an yi wa ƙungiyoyin da ba na kansu ba lakabi da BLASTN (bayanan bayanai na nt NCBI, ƙimar e < 1e−10 da 60% homology). A lokaci guda, an yi wa ƙungiyoyin da ba na kansu ba lakabi da BLASTN (bayanan bayanai na nt NCBI, ƙimar e < 1e−10 da 60% homology). Параллельно чужеродные групповые контиги были аннотированы с помощью BLASTN гомология 60%). A lokaci guda, an yi wa ƙungiyoyin ƙasashen waje lakabi da BLASTN (bayanan bayanai na 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. <1e-10 da гомология 60%). A lokaci guda, an yi wa ƙungiyoyin da ba sa son kansu lakabi da BLASTN (bayanan bayanai na nt NCBI, ƙimar e <1e-10 da 60% homology). An kuma gudanar da BLASTX akan abubuwan da ba na kai ba ta amfani da bayanan NCBI na furotin nr da RefSeq (ƙimar e < 1e−10 da 60% homology). An kuma gudanar da BLASTX akan abubuwan da ba na kai ba ta amfani da bayanan NCBI na furotin nr da RefSeq (ƙimar e < 1e−10 da 60% homology). BLASTX был проведен на несамостоятельных контигах с использованием баз данных белка nr и RefSeq NCBI0 гомология 60%). An kuma yi BLASTX akan cututtukan da ba na kai ba ta amfani da bayanan furotin na nr da RefSeq NCBI (ƙimar e < 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 белка nr и RefSeq NCBI гомология 60%). An kuma yi BLASTX akan cututtukan da ba na kai ba ta amfani da bayanan furotin na nr da RefSeq NCBI (ƙimar e <1e-10 da 60% homology).BLASTN da BLASTX na ƙwayoyin cuta marasa kai suna wakiltar ƙwayoyin cuta na ƙarshe (duba fayil ɗin Ƙarin).
An jera firam ɗin da aka yi amfani da su don PCR a cikin Tebur S1. An yi amfani da Taq DNA polymerase (Bio Basic Canada, Markham, ON) don haɓaka kwayoyin halittar da aka yi niyya don ccfDNA. An yi amfani da waɗannan yanayin amsawa: denaturation a 95°C na minti 3, 95°C na minti 1, saita zafin jiki na annealing na minti 1, tsawaitawa a 72°C na minti 1, zagayowar 35, kuma a ƙarshe 72°C cikin mintuna 10. An raba samfuran PCR ta hanyar electrophoresis a cikin gels na agarose (1.5%) wanda ke ɗauke da SYBRTM Safe DNA Gel Stain (Invitrogen, Burlington, ON, Kanada) a 95 V.
An haɗa ƙwayoyin halittar (Mytilus spp.) a cikin ruwan teku mai iskar oxygen (32 PSU) na tsawon awanni 24 a zafin jiki na 4°C. An ƙara ƙwayar halittar plasmid DNA mai ɗauke da wani abu da ke ƙunshe da jerin galectin-7 na ɗan adam (lambar shiga ta NCBI L07769) a cikin kwalbar a wani taro na ƙarshe na 190 μg/μl. Ƙwayoyin halittar da aka ƙera a ƙarƙashin yanayi iri ɗaya ba tare da ƙara DNA ba sune masu sarrafawa. Tankin sarrafawa na uku ya ƙunshi DNA ba tare da ƙwayoyin halittar halittar ba. Don sa ido kan ingancin DNA a cikin ruwan teku, an ɗauki samfuran ruwan teku (20 μl; maimaitawa uku) daga kowane tanki a lokacin da aka ƙayyade. Don gano ƙwayoyin halittar DNA na plasmid, an girbe ƙwayoyin halittar LB a lokutan da aka ƙayyade kuma an yi nazari da qPCR da ddPCR. Saboda yawan gishirin da ke cikin ruwan teku, an narkar da ƙwayoyin halittar a cikin ruwan PCR mai inganci (1:10) kafin duk gwaje-gwajen PCR.
An yi amfani da tsarin BioRad QX200 (Mississauga, Ontario, Kanada). Yi amfani da bayanin yanayin zafi don tantance mafi kyawun zafin jiki (Table S1). An samar da digo ta amfani da janareta mai saukar QX200 (BioRad). An gudanar da ddPCR kamar haka: 95°C na minti 5, zagaye 50 na 95°C na daƙiƙa 30 da kuma zafin jiki mai rage zafi na minti 1 da 72°C na daƙiƙa 30, 4°C na daƙiƙa 5 da 90°C cikin mintuna 5. An auna adadin digo da kuma sakamako masu kyau (adadin kwafi/µl) ta amfani da na'urar karanta digo na QX200 (BioRad). An ƙi samfuran da ke da digo ƙasa da 10,000. Ba a yi amfani da sarrafa tsari a duk lokacin da aka gudanar da ddPCR ba.
An yi amfani da Rotor-Gene® 3000 (Corbett Research, Sydney, Australia) da kuma takamaiman firam ɗin LGALS7. An yi duk PCRs masu ƙima a cikin 20 µl ta amfani da QuantiFast SYBR Green PCR Kit (QIAGEN). An fara qPCR da sakawa na minti 15 a 95°C sannan aka sake yin zagaye 40 a 95°C na daƙiƙa 10 da kuma a 60°C na daƙiƙa 60 tare da tattara bayanai ɗaya. An samar da lanƙwasa na narkewa ta amfani da ma'auni a jere a 95°C na daƙiƙa 5, 65°C na daƙiƙa 60, da kuma 97°C a ƙarshen qPCR. An yi kowane qPCR a cikin sau uku, sai dai samfuran sarrafawa.
Tunda an san mussels da yawan tacewa, da farko mun bincika ko za su iya tacewa da kuma riƙe gutsuttsuran DNA da ke cikin ruwan teku. Mun kuma yi sha'awar ko waɗannan gutsuttsuran sun taru a cikin tsarin lymphatic ɗinsu na rabin-buɗe. Mun warware wannan batu ta hanyar gwaji ta hanyar bin diddigin makomar gutsuttsuran DNA masu narkewa da aka ƙara a cikin tankunan mussel masu launin shuɗi. Don sauƙaƙe bin diddigin gutsuttsuran DNA, mun yi amfani da DNA na plasmid na waje (ba na kai ba) wanda ke ɗauke da kwayar halittar galectin-7 ta ɗan adam. ddPCR ya bin diddigin 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 daidaito a kan lokaci (har zuwa kwanaki 7) ba tare da mussels ba, to a gaban mussels wannan matakin kusan ya ɓace gaba ɗaya cikin awanni 8 (Hoto na 1a,b). An gano gutsuttsuran DNA na waje cikin sauƙi cikin mintuna 15 a cikin ruwan intravalvular da hemolymph (Hoto na 1c). Har yanzu ana iya gano waɗannan gutsuttsuran har zuwa awanni 4 bayan fallasa. Wannan aikin tacewa dangane da gutsuttsuran DNA yana kama da aikin tace ƙwayoyin cuta da algae [31]. Waɗannan sakamakon sun nuna cewa ƙwayoyin halittar mussels na iya tacewa da kuma tara ƙwayoyin halittar DNA na waje a cikin sassan ruwansu.
Yawan DNA na plasmid a cikin ruwan teku a gaban (A) ko rashin (B) na mussels, wanda aka auna ta hanyar ddPCR. A cikin A, an bayyana sakamakon a matsayin kashi-kashi, tare da iyakokin akwatunan suna wakiltar kashi-kashi na 75 da 25. An nuna lanƙwasa logarithmic da aka sanya a ja, kuma yankin da aka yi wa inuwa da launin toka yana wakiltar tazara ta amincewa ta 95%. A cikin B, layin ja yana wakiltar matsakaicin kuma layin shuɗi yana wakiltar tazara ta amincewa ta 95% don yawan taro. C Tarin DNA na plasmid a cikin hemolymph da ruwan valvular na mussels a lokuta daban-daban bayan ƙara DNA na plasmid. Ana gabatar da sakamakon a matsayin cikakken kwafi da aka gano/mL (±SE).
Na gaba, mun binciki asalin ccfDNA a cikin mussels da aka tattara daga gadajen mussel a Tsibirin Kerguelen, wani rukuni mai nisa na tsibirai masu ƙarancin tasirin ɗan adam. Don wannan dalili, an ware cccDNA daga mussel hemolymphs kuma an tsarkake ta ta hanyoyin da aka saba amfani da su don tsarkake cccDNA na ɗan adam [32, 33]. Mun gano cewa matsakaicin yawan hemolymph ccfDNA a cikin mussels yana cikin ƙananan micrograms a kowace ml na hemolymph (duba Tebur S2, Ƙarin Bayani). Wannan kewayon yawan ya fi girma fiye da na mutanen da ke da lafiya (ƙarancin nanograms a kowace millilita), amma a lokuta da ba kasafai ba, a cikin marasa lafiya da ke fama da cutar kansa, matakin ccfDNA na iya kaiwa micrograms da yawa a kowace millilita [34, 35]. Binciken girman rarrabawar hemolymph ccfDNA ya nuna cewa waɗannan gutsuttsuran sun bambanta sosai a girma, tun daga 1000 bp zuwa 1000 bp. har zuwa 5000 bp (Hoto na 2). An samu irin wannan sakamako ta amfani da Kit ɗin Binciken QIAamp da ke tushen silica, wata hanya da aka saba amfani da ita a kimiyyar binciken laifuka don ware da tsarkake DNA na kwayoyin halitta cikin sauri daga samfuran DNA masu ƙarancin yawan amfani, gami da ccfDNA [36].
Wakilin ccfDNA electrophoregram na mussel hemolymph. An cire shi da NucleoSnap Plasma Kit (sama) da kuma QIAamp DNA Investigator Kit. B Violin plant yana nuna rarrabawar hemolymph ccfDNA mai yawa (±SE) a cikin mussels. Layukan baƙi da ja suna wakiltar matsakaicin da na farko da na uku, bi da bi.
Kimanin kashi 1% na ccfDNA a cikin mutane da birai suna da tushen ƙasashen waje [21, 37]. Ganin tsarin zagayawar jini na bivalves mai buɗewa, ruwan teku mai wadataccen ƙwayoyin cuta, da kuma girman rarrabawar mussel ccfDNA, mun yi hasashen cewa mussel hemolymph ccfDNA na iya ƙunsar tarin DNA mai yawa da bambancin ƙwayoyin cuta. Don gwada wannan hasashe, mun tsara jerin hemolymph ccfDNA daga samfuran Aulacomya atra da aka tattara daga Tsibirin Kerguelen, suna samar da sama da karanta miliyan 10, 97.6% daga cikinsu sun wuce ikon sarrafa inganci. Daga nan aka rarraba karatun bisa ga tushen kai da wanda ba kai ba ta amfani da bayanan BLASTN da NCBI bivalve (Hoto na S1, Ƙarin Bayani).
A cikin mutane, ana iya sakin DNA na nukiliya da na mitochondrial cikin jini [38]. Duk da haka, a cikin wannan binciken, ba zai yiwu a bayyana dalla-dalla DNA na kwayar halittar mussels na nukiliya ba, ganin cewa ba a tsara ko bayyana kwayar halittar A. atra ba. Duk da haka, mun sami damar gano wasu gutsuttsuran ccfDNA na asalinmu ta amfani da ɗakin karatu na bivalve (Hoto na S2, Ƙarin Bayani). Mun kuma tabbatar da kasancewar gutsuttsuran DNA na asalinmu ta hanyar haɓaka PCR na waɗannan kwayoyin halittar A. atra waɗanda aka tsara (Hoto na 3). Hakazalika, ganin cewa kwayar halittar mitochondrial na A. atra tana samuwa a cikin bayanan jama'a, mutum zai iya samun shaida don kasancewar gutsuttsuran ccfDNA na mitochondrial a cikin hemolymph na A. atra. An tabbatar da kasancewar gutsuttsuran DNA na mitochondrial ta hanyar haɓaka PCR (Hoto na 3).
Akwai nau'ikan kwayoyin halitta daban-daban na mitochondrial a cikin hemolymph na A. atra (dige ja - lambar hannun jari: SRX5705969) da M. platensis (dige shuɗi - lambar hannun jari: SRX5705968) wanda PCR ta ƙara. Hoton da aka daidaita daga Breton et al., 2011 B Ƙara girman jinin haemoglobin daga A. atra An adana shi akan takardar FTA. Yi amfani da naushi mai girman mm 3 don ƙara kai tsaye zuwa bututun PCR wanda ke ɗauke da haɗin PCR.
Ganin yawan ƙwayoyin cuta a cikin ruwan teku, da farko mun mayar da hankali kan siffanta jerin DNA na ƙwayoyin cuta a cikin hemolymph. Don yin wannan, muna amfani da dabaru biyu daban-daban. Dabaru na farko ya yi amfani da Kraken2, wani shiri na rarraba jerin abubuwa bisa algorithm wanda zai iya gano jerin ƙwayoyin cuta tare da daidaito daidai da BLAST da sauran kayan aiki [28]. An tabbatar da cewa fiye da 6719 sun samo asali ne daga ƙwayoyin cuta, yayin da 124 da 64 sun samo asali ne daga archaea da ƙwayoyin cuta, bi da bi (Hoto na 4). Guraben DNA na ƙwayoyin cuta mafi yawa sune Firmicutes (46%), Proteobacteria (27%), da Bacteroidetes (17%) (Hoto na 4a). Wannan rarrabawar ta yi daidai da binciken da aka yi a baya na microbiome na mussel blue na teku [39, 40]. Gammaproteobacteria sune babban aji na Proteobacteria (44%), gami da Vibrionales da yawa (Hoto na 4b). Hanyar ddPCR ta tabbatar da kasancewar gutsuttsuran DNA na Vibrio a cikin ccfDNA na A. atra hemolymph (Hoto na 4c) [41]. Domin samun ƙarin bayani game da asalin ƙwayoyin cuta na ccfDNA, an ɗauki ƙarin hanyar (Hoto na S2, Ƙarin Bayani). A wannan yanayin, an haɗa karatun da aka haɗa a matsayin karatun da aka haɗa a gefe guda kuma an rarraba su azaman na kai (bivalves) ko asalin kai ba ta amfani da BLASTN da ƙimar e na 1e−3 da kuma yankewa tare da daidaiton >90%. A wannan yanayin, an haɗa karatun da aka haɗa a matsayin karatun da aka haɗa a gefe guda kuma an rarraba su azaman na kai (bivalves) ko asalin kai ba ta amfani da BLASTN da ƙimar e na 1e−3 da kuma yankewa tare da daidaiton >90%. Видео (двустворчатые моллюски) или чужие по происхождению A wannan yanayin, an tattara karatun da suka yi karo da juna a matsayin karatun da aka haɗa biyu kuma an rarraba su azaman na asali (bivalve) ko wanda ba na asali ba ta amfani da BLASTN da ƙimar e na 1e-3 kuma an yanke su da daidaiton >90%.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%同源性的截止值分类为自身（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 ，同源性 的 分类 自身 （双 壳类） 非 自身。 Видео (двустворчатые моллюски) или несобственные по происхождению с использованием значений e BLASTN и 1e-3 A wannan yanayin, an tattara karatun da aka haɗa a matsayin karatun da aka haɗa biyu kuma an rarraba su azaman nasu (bivalves) ko waɗanda ba na asali ba ta amfani da ƙimar e BLASTN da 1e-3 ​​kuma an ƙayyade iyakar daidaitawar >90%.Tunda har yanzu ba a tsara jerin kwayoyin halittar A. atra ba, mun yi amfani da dabarun haɗa kwayoyin halitta na MEGAHIT Next Generation Sequencing (NGS). An gano jimillar kwayoyin halitta 147,188 a matsayin tushen asali (bivalves). An fashe waɗannan kwayoyin halitta da ƙimar e-10 ta amfani da BLASTN da BLASTX. Wannan dabarar ta ba mu damar gano gutsuttsuran DNA guda 482 waɗanda ba su da bivalve a cikin A. atra ccfDNA. Fiye da rabi (57%) na waɗannan gutsuttsuran DNA an samo su ne daga ƙwayoyin cuta, galibi daga gill symbionts, gami da sulfotrophic symbionts, da kuma daga gill symbionts Solemya velum (Hoto na 5).
Yawa mai alaƙa a matakin nau'in. B Bambancin ƙwayoyin cuta na manyan ƙwayoyin cuta guda biyu (Firmicutes da Proteobacteria). Wakiltar ƙaruwar ddPCR C Vibrio spp. A. Rarrabuwar kwayar halittar 16S rRNA (shuɗi) a cikin hemolymphs uku na atra.
An yi nazarin jimillar ƙwayoyin cuta guda 482 da aka tattara. Bayanin gabaɗaya game da rarrabawar taxonomic na bayanan metagenomic contig (prokaryotes da eukaryotes). B Cikakken rarrabawar gutsuttsuran DNA na ƙwayoyin cuta da BLASTN da BLASTX suka gano.
Binciken Kraken2 ya kuma nuna cewa mussel ccfDNA ya ƙunshi gutsuttsuran DNA na archaeal, gami da gutsuttsuran DNA na Euryarchaeota (65%), Crenarchaeota (24%), da Thaurmarcheota (11%) (Hoto na 6a). Kasancewar gutsuttsuran DNA da aka samo daga Euryarchaeota da Crenarchaeota, waɗanda a da ake samu a cikin al'ummar ƙwayoyin cuta na mussels na California, bai kamata ya zama abin mamaki ba [42]. Kodayake Euryarchaeota galibi ana danganta shi da yanayi mai tsauri, yanzu an gane cewa duka Euryarchaeota da Crenarcheota suna cikin prokaryotes da aka fi sani a cikin yanayin cryogenic na teku [43, 44]. Kasancewar ƙwayoyin cuta na methanogenic a cikin mussels ba abin mamaki bane, idan aka yi la'akari da rahotannin baya-bayan nan na yawan ɗigon methane daga ɗigon ƙasa a kan tudun Kerguelen [45] da kuma yiwuwar samar da methane na ƙwayoyin cuta da aka lura a bakin tekun Tsibirin Kerguelen [46].
Daga nan hankalinmu ya koma ga karatu daga ƙwayoyin cuta na DNA. A gwargwadon iliminmu, wannan shine bincike na farko da ba a yi niyya ba game da abubuwan da ke cikin ƙwayoyin cuta na mussels. Kamar yadda aka zata, mun sami gutsuttsuran ƙwayoyin cuta na DNA (Caudovirales) (Hoto na 6b). Duk da haka, DNA mafi yawan ƙwayoyin cuta ya fito ne daga phylum na ƙwayoyin cuta na nucleocytoviruses, wanda aka fi sani da ƙwayar cutar DNA mai girma ta nuclear cytoplasmic (NCLDV), wacce ke da mafi girman kwayar halittar kowace ƙwayar cuta. A cikin wannan phylum, yawancin jerin DNA sun kasance na iyalan Mimimidovaridae (58%) da Poxviridae (21%), waɗanda masu masaukin su na halitta sun haɗa da ƙasusuwa da arthropods, yayin da ƙaramin rabo na waɗannan jerin DNA ya kasance na algae na virological da aka sani. Yana kamuwa da algae na eukaryotic na ruwa. An kuma samo jerin daga ƙwayar cutar Pandora, babbar ƙwayar cuta mai girman kwayar halitta mafi girma fiye da kowace kwayar cuta da aka sani. Abin sha'awa, yawan masu masaukin baki da aka sani da kamuwa da cutar, kamar yadda aka ƙayyade ta hanyar jerin kwayoyin halittar hemolymph ccfDNA, ya kasance babba (Hoto na S3, Ƙarin Bayani). Ya haɗa da ƙwayoyin cuta waɗanda ke kamuwa da kwari kamar Baculoviridae da Iridoviridae, da kuma ƙwayoyin cuta waɗanda ke kamuwa da amoeba, algae da ƙashi. Mun kuma sami jerin da suka dace da kwayar halittar Pithovirus sibericum. An fara ware ƙwayoyin cuta na Pitovirus (wanda aka fi sani da "ƙwayoyin cuta na zombie") daga permafrost mai shekaru 30,000 a Siberia [47]. Don haka, sakamakonmu ya yi daidai da rahotannin da suka gabata wanda ke nuna cewa ba duk nau'ikan waɗannan ƙwayoyin cuta na zamani ba ne suka mutu [48] kuma waɗannan ƙwayoyin cuta na iya kasancewa a cikin yanayin halittu na ruwa na subarctic.
A ƙarshe, mun gwada don ganin ko za mu iya samun gutsuttsuran DNA daga wasu dabbobi masu ƙwayoyin halitta da yawa. An gano jimillar ƙwayoyin cuta na ƙasashen waje guda 482 ta hanyar BLASTN da BLASTX tare da ɗakunan karatu na nt, nr da RefSeq (genomic da protein). Sakamakonmu ya nuna cewa daga cikin gutsuttsuran ccfDNA na dabbobin da ke da ƙwayoyin halitta da yawa DNA na ƙasusuwan ƙashi ya fi rinjaye (Hoto na 5). An kuma gano gutsuttsuran DNA daga kwari da sauran nau'ikan. Ba a gano wani babban ɓangare na gutsuttsuran DNA ba, wataƙila saboda ƙarancin wakilcin nau'ikan halittu na ruwa a cikin bayanan kwayoyin halitta idan aka kwatanta da nau'ikan halittu na ƙasa [49].
A cikin wannan takarda, mun yi amfani da ra'ayin LB ga mussels, muna jayayya cewa jerin harbe-harben hemolymph ccfDNA na iya ba da haske game da abubuwan da ke cikin yanayin halittu na bakin teku na teku. Musamman, mun gano cewa 1) mussel hemolymph ya ƙunshi babban taro (matakan microgram) na manyan gutsuttsuran DNA masu yawo (~1-5 kb); 2) waɗannan gutsuttsuran DNA duka masu zaman kansu ne kuma ba masu zaman kansu ba ne 3) Daga cikin hanyoyin ƙasashen waje na waɗannan gutsuttsuran DNA, mun sami ƙwayoyin cuta, archaeal da viral DNA, da kuma DNA na sauran dabbobi masu yawa; 4) Tarin waɗannan gutsuttsuran ccfDNA na ƙasashen waje a cikin hemolymph yana faruwa da sauri kuma yana ba da gudummawa ga aikin tacewa na ciki na mussels. A ƙarshe, bincikenmu ya nuna cewa ra'ayin LB, wanda zuwa yanzu aka yi amfani da shi galibi a fannin biomedicine, yana ɓoye tushen ilimi mai wadata amma wanda ba a bincika ba wanda za a iya amfani da shi don fahimtar hulɗar da ke tsakanin nau'ikan sentinel da muhallinsu.
Baya ga birai, an ruwaito cewa an ware ccfDNA a cikin dabbobi masu shayarwa, ciki har da beraye, karnuka, kuliyoyi, da dawakai [50, 51, 52]. Duk da haka, gwargwadon iliminmu, bincikenmu shine na farko da ya ba da rahoton gano da kuma jerin ccfDNA a cikin nau'ikan ruwa tare da tsarin zagayawa a buɗe. Wannan fasalin jiki da ikon tace mussels na iya, aƙalla a wani ɓangare, bayyana halaye daban-daban na girman gutsuttsuran DNA idan aka kwatanta da sauran nau'ikan. A cikin mutane, yawancin gutsuttsuran DNA da ke yawo a cikin jini ƙananan gutsuttsura ne waɗanda suka kama daga 150 zuwa 200 bp. tare da matsakaicin kololuwar 167 bp [34, 53]. Ƙaramin yanki amma mai mahimmanci na gutsuttsuran DNA suna tsakanin 300 zuwa 500 bp a girma, kuma kusan 5% sun fi 900 bp tsayi. [54]. Dalilin wannan rarraba girman shine babban tushen ccfDNA a cikin jini yana faruwa ne sakamakon mutuwar ƙwayoyin halitta, ko dai saboda mutuwar ƙwayoyin halitta ko kuma saboda necrosis na ƙwayoyin hematopoietic da ke yawo a cikin mutane masu lafiya ko kuma saboda apoptosis na ƙwayoyin ƙari a cikin marasa lafiya da ke fama da cutar kansa (wanda aka sani da zagayawa da ƙari DNA). , ctDNA). Girman rarraba hemolymph ccfDNA da muka samu a cikin ƙwayoyin halitta ya kasance daga 1000 zuwa 5000 bp, yana nuna cewa mussel ccfDNA yana da asali daban. Wannan hasashe ne mai ma'ana, tunda mussels suna da tsarin jijiyoyin jini na rabin-buɗe kuma suna rayuwa a cikin yanayin ruwa na ruwa wanda ke ɗauke da yawan ƙwayoyin halittar DNA na ƙwayoyin cuta. A zahiri, gwaje-gwajen dakin gwaje-gwajenmu ta amfani da DNA na waje sun nuna cewa mussels suna tara gutsuttsuran DNA a cikin ruwan teku, aƙalla bayan 'yan awanni suna lalacewa bayan shan ƙwayoyin halitta da/ko sake su da/ko adana su a cikin ƙungiyoyi daban-daban. Ganin ƙarancin ƙwayoyin halitta (duka prokaryotic da eukaryotic), amfani da sassan da ke cikin jijiyoyin jini zai rage adadin ccfDNA daga tushen kansu da kuma daga kafofin waje. Idan aka yi la'akari da mahimmancin garkuwar jiki ta bivalve da kuma yawan ƙwayoyin halitta masu yawo, mun ƙara hasashen cewa ko da ccfDNA na ƙasashen waje yana da wadata a cikin ƙwayoyin halitta masu yawo waɗanda ke tara DNA na ƙasashen waje lokacin da aka sha ƙwayoyin cuta da/ko tarkacen ƙwayoyin halitta. Idan aka haɗa su, sakamakonmu ya nuna cewa bivalve hemolymph ccfDNA wani ma'ajiyar bayanai ne na musamman na kwayoyin halitta kuma yana ƙarfafa matsayinsu a matsayin nau'in sentinel.
Bayananmu sun nuna cewa yin bincike da kuma nazarin gutsuttsuran ƙwayoyin cuta na hemolymph ccfDNA da aka samo daga ƙwayoyin cuta na iya samar da muhimman bayanai game da ƙwayoyin cuta masu masaukin baki da ƙwayoyin cuta da ke cikin yanayin halittu na ruwa da ke kewaye. Dabaru na yin bincike da harbe-harbe sun bayyana jerin ƙwayoyin cuta na commensal A. atra gill waɗanda da ba a rasa ba idan an yi amfani da hanyoyin gano 16S rRNA na gargajiya, saboda wani ɓangare na son zuciya a ɗakin karatu. A gaskiya ma, amfani da bayanan LB da muka tattara daga M. platensis a cikin wannan layin mussel a Kerguelen ya nuna cewa abun da ke cikin symbionts na ƙwayoyin cuta da ke da alaƙa da gill iri ɗaya ne ga nau'ikan mussel guda biyu (Hoto na S4, Ƙarin Bayani). Wannan kamanceceniya na mussels guda biyu daban-daban na halitta na iya nuna tsarin al'ummomin ƙwayoyin cuta a cikin ma'ajiyar sanyi, sulfurous, da kuma aman wuta na Kerguelen [55, 56, 57, 58]. An bayyana manyan matakan ƙwayoyin cuta masu rage sulfur sosai lokacin girbe mussels daga yankunan bakin teku masu turbated [59], kamar bakin tekun Port-au-France. Wata yiwuwar kuma ita ce yaduwar ƙwayoyin halittar mussel na iya shafar ƙwayoyin halittar mussel na commensal [60, 61]. Ana buƙatar ƙarin bincike don tantance alaƙar da ke tsakanin yanayin ruwa, saman ƙasan teku, da kuma abubuwan da ke cikin ƙwayoyin halittar da ke cikin mussels. Waɗannan nazarin suna gudana a halin yanzu.
Tsawon da yawan sinadarin hemolymph ccfDNA, sauƙin tsarkakewarsa, da kuma inganci mai kyau don ba da damar saurin jerin bindigogi su ne wasu daga cikin fa'idodi da yawa na amfani da mussel ccfDNA don tantance bambancin halittu a cikin yanayin halittu na bakin teku. Wannan hanyar tana da tasiri musamman don siffanta al'ummomin ƙwayoyin cuta (viromes) a cikin wani yanayi na halitta [62, 63]. Ba kamar ƙwayoyin cuta, archaea, da eukaryotes ba, kwayoyin halittar ƙwayoyin cuta ba su ƙunshi kwayoyin halittar da aka kiyaye su ta hanyar phylogenetic kamar jerin 16S. Sakamakonmu ya nuna cewa ana iya amfani da biopsy na ruwa daga nau'ikan alamu kamar mussels don gano adadi mai yawa na gutsuttsuran ƙwayoyin cuta na ccfDNA da aka sani suna kamuwa da masu masaukin baki waɗanda galibi ke zaune a cikin yanayin halittu na bakin teku. Wannan ya haɗa da ƙwayoyin cuta da aka sani suna kamuwa da protozoa, arthropods, kwari, tsire-tsire, da ƙwayoyin cuta na ƙwayoyin cuta (misali, bacteriophages). An sami irin wannan rarrabawa lokacin da muka bincika virome na hemolymph ccfDNA na mussels masu launin shuɗi (M. platensis) da aka tattara a cikin wannan layin mussel a Kerguelen (Tebur S2, Ƙarin Bayani). Jerin bindigogi na ccfDNA hakika sabuwar hanya ce da ke ƙara samun ci gaba a cikin nazarin virome na mutane ko wasu nau'ikan halittu [21, 37, 64]. Wannan hanyar tana da amfani musamman don nazarin ƙwayoyin cuta na DNA masu nau'ikan biyu, tunda babu kwayar halitta ɗaya da aka kiyaye a tsakanin dukkan ƙwayoyin cuta na DNA masu nau'ikan biyu, wanda ke wakiltar nau'in ƙwayoyin cuta mafi bambancin da faɗi a Baltimore [65]. Kodayake yawancin waɗannan ƙwayoyin cuta ba su kasance a cikin rarrabuwa ba kuma suna iya haɗawa da ƙwayoyin cuta daga wani ɓangare na duniyar ƙwayoyin cuta da ba a sani ba gaba ɗaya [66], mun gano cewa viromes da jeri na mussels A. atra da M. platensis suna faɗuwa tsakanin nau'ikan biyu. haka nan (duba hoto na S3, ƙarin bayani). Wannan kamanceceniya ba abin mamaki bane, domin yana iya nuna rashin zaɓi a cikin ɗaukar DNA da ke cikin muhalli. Ana buƙatar nazarin da za a yi nan gaba ta amfani da RNA mai tsabta a halin yanzu don tantance ƙwayar RNA.
A cikin bincikenmu, mun yi amfani da wani bututu mai tsauri wanda aka daidaita daga aikin Kowarski da abokan aikinsa [37], waɗanda suka yi amfani da sharewar matakai biyu na karatu da abubuwan da suka shafi kafin da bayan haɗa ccfDNA na asali, wanda ya haifar da babban kaso na karatu da ba a tsara ba. Saboda haka, ba za mu iya kawar da cewa wasu daga cikin waɗannan karatun da ba a tsara ba har yanzu suna da asalinsu, musamman saboda ba mu da kwayar halittar da ke nuna wannan nau'in mussel. Mun kuma yi amfani da wannan bututun saboda mun damu da chimeras tsakanin karatun kai da na kai da kuma tsawon karatun da Illumina MiSeq PE75 ke samarwa. Wani dalili kuma na yawancin karatun da ba a tsara ba shine cewa yawancin ƙwayoyin cuta na ruwa, musamman a wurare masu nisa kamar Kerguelen, ba a yi musu bayanin komai ba. Mun yi amfani da Illumina MiSeq PE75, muna ɗauka cewa tsawon gutsuttsuran ccfDNA yayi kama da na ɗan adam ccfDNA. Don nazarin da za a yi nan gaba, idan aka yi la'akari da sakamakonmu da ke nuna cewa hemolymph ccfDNA yana da tsawon karatu fiye da mutane da/ko dabbobi masu shayarwa, muna ba da shawarar amfani da dandamalin jerin abubuwa wanda ya fi dacewa da gutsuttsuran ccfDNA masu tsayi. Wannan aikin zai sauƙaƙa gano ƙarin alamu don zurfafa bincike. Samun cikakken jerin kwayoyin halittar nukiliya na A. atra wanda ba a samu a halin yanzu zai kuma sauƙaƙa wariya ga ccfDNA daga tushen kai da waɗanda ba na kai ba. Ganin cewa bincikenmu ya mayar da hankali kan yiwuwar 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 haɓaka sabbin kayan aiki da bututun mai don ƙara ƙarfin wannan hanyar don nazarin bambancin ƙwayoyin cuta na mussels. yanayin halittu na teku.
A matsayin wani abu mai nuna alamun cutar asibiti wanda ba ya yin illa ga lafiya, hauhawar matakan ccfDNA a cikin jini na ɗan adam yana da alaƙa da cututtuka daban-daban, lalacewar nama, da yanayin damuwa [67,68,69]. Wannan ƙaruwa yana da alaƙa da sakin gutsuttsuran DNA na asalinsa bayan lalacewar nama. Mun magance wannan batu ta amfani da matsanancin zafi, inda mussels suka ɗan fallasa su ga zafin jiki na 30 °C na ɗan lokaci. Mun yi wannan bincike kan nau'ikan mussels guda uku daban-daban a cikin gwaje-gwaje uku masu zaman kansu. Duk da haka, ba mu sami wani canji a matakan ccfDNA ba bayan matsanancin zafi (duba Hoto na S5, ƙarin bayani). Wannan binciken na iya bayyana, aƙalla a wani ɓangare, gaskiyar cewa mussels suna da tsarin zagayawa na rabin-buɗe kuma suna tara adadi mai yawa na DNA na waje saboda yawan aikin tace su. A gefe guda kuma, mussels, kamar sauran marasa ƙashi da yawa, na iya zama masu juriya ga lalacewar nama da damuwa ke haifarwa, ta haka ne ke iyakance sakin ccfDNA a cikin hemolymph ɗinsu [70, 71].
Zuwa yanzu, nazarin DNA na bambancin halittu a cikin yanayin halittu na ruwa ya fi mayar da hankali kan tsarin DNA na muhalli (eDNA). Duk da haka, wannan hanyar yawanci tana da iyaka a cikin nazarin bambancin halittu lokacin da ake amfani da firam. Amfani da jerin bindigogi yana keta iyakokin PCR da zaɓin saitin firam ɗin da ba daidai ba. Don haka, a wata ma'ana, hanyarmu ta fi kusa da hanyar jerin eDNA Shotgun mai ƙarfi da aka yi amfani da ita kwanan nan, wacce ke iya tsara DNA da aka raba kai tsaye da kuma nazarin kusan dukkan halittu [72, 73]. Duk da haka, akwai wasu batutuwa na asali da suka bambanta LB daga hanyoyin eDNA na yau da kullun. Tabbas, babban bambanci tsakanin eDNA da LB shine amfani da masu masaukin matatun halitta. An ba da rahoton amfani da nau'ikan ruwa kamar soso da bivalves (Dresseina spp.) azaman matattarar halitta don nazarin eDNA [74, 75]. Duk da haka, binciken Dreissena ya yi amfani da biopsies na nama wanda aka cire DNA daga ciki. Binciken ccfDNA daga LB ba ya buƙatar biopsy na nama, kayan aiki na musamman kuma wani lokacin tsada da dabaru da ke da alaƙa da eDNA ko biopsy na nama. A gaskiya ma, kwanan nan mun ruwaito cewa ana iya adana ccfDNA daga LB kuma a yi nazari da shi tare da tallafin FTA ba tare da kula da sarkar sanyi ba, wanda babban ƙalubale ne ga bincike a yankuna masu nisa [76]. Cire ccfDNA daga biopsy na ruwa shi ma mai sauƙi ne kuma yana ba da ingantaccen DNA don jerin bindigogi da nazarin PCR. Wannan babban fa'ida ne idan aka yi la'akari da wasu ƙuntatawa na fasaha da ke da alaƙa da nazarin eDNA [77]. Sauƙin da ƙarancin kuɗin hanyar ɗaukar samfur suma sun dace musamman don shirye-shiryen sa ido na dogon lokaci. Baya ga babban ƙarfin tace su, wani sanannen fasalin bivalves shine sinadaran mucopolysaccharide na maƙogwaronsu, wanda ke haɓaka shan ƙwayoyin cuta [78, 79]. Wannan ya sa bivalves ya zama matattarar halitta mai kyau don siffanta bambancin halittu da tasirin sauyin yanayi a cikin yanayin halittu na ruwa. Duk da cewa kasancewar gutsuttsuran DNA da aka samo daga mai masaukin baki ana iya ganin su a matsayin iyakancewar hanyar idan aka kwatanta da eDNA, farashin da ke tattare da samun irin wannan ccfDNA na asali idan aka kwatanta da eDNA a lokaci guda ana iya fahimta shi don yawan bayanai da ake da su don nazarin lafiya. offset host. Wannan ya haɗa da kasancewar jerin ƙwayoyin cuta da aka haɗa cikin kwayar halittar mai masaukin baki. Wannan yana da mahimmanci musamman ga mussels, idan aka yi la'akari da kasancewar ƙwayoyin cutar leukemic da aka watsa a kwance a cikin bivalves [80, 81]. Wata fa'idar LB fiye da eDNA ita ce tana amfani da aikin phagocytic na ƙwayoyin jini da ke yawo a cikin hemolymph, wanda ke cinye 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 zagayawa na kwana biyu, wanda ke ƙara haɗar yadudduka daban-daban na ruwan teku, yana ba da damar kama eDNA iri ɗaya. [83, 84]. Saboda haka, nazarin mussel ccfDNA hanya ce mai ban sha'awa idan aka yi la'akari da tasirin abinci mai gina jiki, tattalin arziki, da muhalli na mussels. Kamar nazarin LB da aka tattara daga mutane, wannan hanyar kuma tana buɗe yiwuwar auna canje-canjen kwayoyin halitta da epigenetic a cikin DNA mai masaukin baki don mayar da martani ga abubuwan waje. Misali, ana iya hasashen fasahar jerin tsararraki na ƙarni na uku don yin nazarin methylation na kwayoyin halitta a cikin ccfDNA na asali ta amfani da jerin nanopore. Ya kamata a sauƙaƙe wannan tsari ta hanyar gaskiyar cewa tsawon gutsuttsuran mussel ccfDNA ya dace da dandamalin jerin da aka karanta na dogon lokaci waɗanda ke ba da damar nazarin methylation na DNA na kwayoyin halitta daga jerin jerin guda ɗaya ba tare da buƙatar canje-canjen sinadarai ba.85,86] Wannan wata dama ce mai ban sha'awa, kamar yadda aka nuna cewa tsarin methylation na DNA yana nuna martani ga damuwar muhalli kuma yana ci gaba tsawon tsararraki da yawa. Saboda haka, yana iya samar da fahimta mai mahimmanci game da hanyoyin da ke ƙarƙashin jagorancin amsawa bayan fallasa ga canjin yanayi ko gurɓatattun abubuwa [87]. Duk da haka, amfani da LB ba shi da iyaka. Ba sai an faɗi ba, wannan yana buƙatar kasancewar nau'ikan alamu a cikin yanayin halittu. Kamar yadda aka ambata a sama, amfani da LB don tantance bambancin halittu na wani yanayi na halitta yana buƙatar ingantaccen bututun bioinformatics wanda ke la'akari da kasancewar gutsuttsuran DNA daga tushen. Wata babbar matsala ita ce samuwar kwayoyin halitta na asali ga nau'ikan ruwa. Ana fatan cewa shirye-shirye kamar Aikin Halittar Dabbobi na Marine da kuma aikin Fish10k da aka kafa kwanan nan [88] zai sauƙaƙa irin wannan bincike a nan gaba. Amfani da ra'ayin LB ga halittu masu ciyar da matatun ruwa shi ma ya dace da sabbin ci gaba a fasahar tsara abubuwa, wanda hakan ya sa ya dace da haɓaka alamun halittu masu yawa-ohm don samar da muhimman bayanai game da lafiyar mazaunin ruwa don mayar da martani ga damuwar muhalli.
An ajiye bayanan jerin kwayoyin halitta a cikin Jerin NCBI Karanta Taskar https://www.ncbi.nlm.nih.gov/sra/SRR8924808 a ƙarƙashin Bioprojects SRR8924808.
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