thanin itsfood alga(Soule 2009).Thus, generalizing negative results obtained from problem-atical data to the rest of the species of kleptoplastic slugs,especiallyinthepresenceofthelargeamountofbiochemicaldata,includingPCR,andshowingthepresenceoftransferredgenes, seem inappropriate.However, another recent report failed to ﬁnd any evi-dence for transferred algal nuclear genes in the transcrip-tome of
, although 19 chloroplast-encodedgene sequences emerged from the analysis (Pelletreauet al. 2011). Unfortunately, while this study investigated
, the transcriptome sequencing yielded
20% (14,000) of the contigs of the
transcrip-tome and was not compared with
native sequen-ces. Thus, the lack of both sequencing depth and databasevoids suggests that rare transcripts could have been easilymissed. Indeed, a conclusion of this analysis was that ‘‘moreexhaustive sequencing may be required’’ to adequatelytest for the presence of transferred genes in
(Pelletreau et al. 2011).In spite of the issues with these negative studies(Pelletreau et al. 2011 ;Wa¨gele et al. 2011), both nonetheless
point to the likelihood that if transferred algal nucleargenes are present in sacoglossan slugs, they will be of verylow copy number, their expression will be low, and knowl-edge of the native algal sequence will facilitate, perhapseven be required for, the annotation. Therefore, insteadof assuming that a large number of transcripts for nuclear-encoded and plastid-encoded proteins would be present inthe slug cell, we have hypothesized that such would be ex-ceedingly rare in the transcriptome and have done our ownanalysis of
creating large amounts of Illumina-generated sequence data. In addition, to facilitate an accu-rate annotation, wehavesequenced the genome of
as well as the algal transcriptome, to provide a databaseof native algal transcript sequences. Our
tran-scriptome data contain a variety, albeit rare, of chloroplast-encoded transcripts and, in addition, rarer still, at least 111reads that match 52 algal nuclear-encoded sequences, in-cluding one that matches exactly an algal nuclear sequencefoundbyPCRin
Materials and Methods
Animals and Algae
were collected from a salt marshnear Menemsha on Martha’s Vineyard, MA, and shipped toTampa, FL. The sea slugs were placed into aerated aquariacontaining artiﬁcial seawater made from Instant Oceansalts dissolved in sterilized deionized water at 1,000 mOsm.The aquaria were kept at 10
C in a cold box equipped withﬂuorescent lights set on a 14:10 light–dark cycle. The slugswere starved for at least 2 months under the foregoingconditions before use in experiments.Filaments of
used in the experiments camefrom a culture that has been maintained in our lab for morethan 10 years. The initial ﬁlament used to establish the cul-ture came from the same marsh that provided the slugs.The
ﬁlaments are grown in a modiﬁed F/2enriched seawater at 250 mOsm in an incubator at20
C, illuminated by ﬂuorescent lights on a 14:10 light–dark cycle (Pierce et al. 1996). Since the original culturewas established from a single ﬁlament and has only grownvegetatively, the ﬁlaments used here are clonal.
Extraction of Genomic DNA from
Genomic DNA was extracted from 10 g of algal ﬁlamentsusing a protocol modiﬁed fromAl-Samarrai and Schmid(2000).The ﬁlaments were rinsed with fresh culture media,blotted to remove excess liquid, frozen in liquid nitrogen,and ground to a powder with a precooled mortar andpestle. Lysis buffer (40 mM Tris–acetate, 20 mM sodiumacetate, 1 mM ethylenediaminetetraacetic acid, and 1%sodium dodecyl sulfate, pH 7.0) was added to the frozenalgal powder and extracted for 10 min at room tempera-ture (RT) while rotating. To facilitate the precipitation of proteins and polysaccharides, 5 M NaCl was added to eachtube (2:5 v/v), the tubes were vortexed and then centri-fuged at 12,000
g at 4
C. The supernatant was extractedwith phenol/chloroform (1:1 v/v) and spun at 10,000
C. The aqueous phase was extracted with chloroform(1:1 v/v) and centrifuged again at 10,000
C. DNAwas precipitated from the aqueous phase by adding isopro-panol (1:1 v/v) and spun at 10,000
C to pellet thenucleic acids. The precipitated DNA was resuspended inlysis buffer containing 100
g/ml RNase A (Qiagen, Valencia,CA) and rotated for 30 min at RT. Following thatincubation, the DNA was run through the puriﬁcationprocess a second time. The ﬁnal precipitated DNA waswashed twicewith 75% ethanol, air dried, resuspended in nu-clease-free water, quantiﬁed spectrophotometrically at 260nm, and then express shipped to BGI in Hong Kong, China.
transcriptome data sets were used in ouranalysis. One set was produced earlier by 454 sequencing(Schwartz et al. 2010) and the second set using the Illuminaplatform as described below. The 454 data are referred to asEST and the Illumina data as RNA-seq, hereafter.
Extraction of RNA from
Total RNA was isolated from about 1 gm of algal ﬁlamentsafter 5 h of exposure to light. The ﬁlaments were groundinto a frozen powder as described above, RNA was ex-tracted using the RNeasy Plant mini kit (Qiagen, Valencia,CA) following the manufacturer’s instructions, quantiﬁedspectrophotometrically at 260 nm, placed on dry ice,and express shipped immediately to BGI in Hong Kong, CN.
Extraction of RNA from
Total RNA was isolated from
2-month starved slugs af-ter 8 h of light exposure by homogenization in Trizol Re-agent (Invitrogen, Carlsbad, CA). The homogenate was
Pierce et al.
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