The cockroach Blattella germanica obtains nitrogen from uric acid
through a metabolic pathway shared with its bacterial endosymbiont.
Rafael Patiño-Navarrete1, MariaDolors Piulachs2, Xavier Belles2, Andrés Moya1, Amparo
Latorre1* and Juli Peretó1,3*
1
InstitutCavanilles de Biodiversitat i Biologia Evolutiva (Universitat de València), C/
Catedràtic José Beltrán nº 2, 46980 Paterna-Spain.
2
Institut de Biologia Evolutiva (Consejo Superior de Investigaciones Científicas and
Universitat Pompeu Fabra), Passeig Marítim de la Barceloneta nº 37-49, 08003 BarcelonaSpain.
3
Departament de Bioquímica i Biologia Molecular, Universitat de València.
Electronic Supplementary Material
Supplementary Materials and Methods
Transcriptome sequencing
Blattella germanica specimens used for the whole transcriptome analysis were obtained from
a population reared at the Institut de Biologia Evolutiva (CSIC-UPF). Conditions were 70%
humidity at 30 ºC and a commercial dog food diet.
The whole transcriptome was analysed in three different tissues, fat body and ovary, which
harbour Blattabacterium, although in the ovary the endosymbionts are only in transit to infect
the embryos, and epidermis, which is a Blattabacterium-free tissue. Fat body and ovaries
were obtained from six adult females, taken from three to five days after moulting to the adult
form, while the epidermis was obtained from the thoracic dorsum of 5 to 6-day-old 6th instar
nymphs. Three animals were used to pool ovary and epithelium, and five were necessary to
pool the fat body. Total RNA was extracted with the GenEluteTM Mammalian Total RNA
Miniprep Kit (Sigma-Aldrich). RNA samples were sent to be sequenced at GATC-Biotech
(Konstanz, Germany), cDNA was synthesized using the Smart cDNA Construction Kit
(Clontech). The first strand was synthesized using an oligo (dT) followed by a cap-primed
second strand synthesis.
The trimmed sequences were cleaned for Blattabacterium by mapping all the reads against
the genome sequence of Blattabacterium str. Bge (Accession numbers NC_013454,
NC_015679), using MEGABLAST (1), with an e-value cut-off of 10-5 and an identity
percentage of 95.
Assembly and annotation
The assembly was performed using MIRA 3.2 with the options ‘denovo, est, accurate, 454
454_SETTINGS -LR:mxti=1 -ED:acc=1 -DP:ure=1’ in MIRA 3.2(2). Finally, the contigs
obtained were annotated with the Blast2GO suite (3)
Experimental diets
The experimental diets were based on those proposed by Mullins and Cochran (4), with some
modifications. Table S1 describes the composition. Adult females were separated from the
colony as soon as they moulted to the adult form, before changing colour. From this moment,
they were fed for 48 h with the control diet, thereafter they were placed on the experimental
diet for another 48 h. On the fourth day after moulting to the adult form, fat body and ovaries
of each animal were removed. After RNA extraction and cDNA synthesis, the relative
expression of all genes encoding for uricolytic enzymes, glutamine synthetase and urease
were measured with Real-Time-qPCR (RT-qPCR). Three biological samples were measured
for each experiment, and three technical replicates were done for each sample. As a reference
for all genes encoded by B. germanica the actin 5c gene (accession no. AJ862721) was used,
while the gene encoding for the Blattabacterium elongation factor EF-TU (accession no.
YP_00328380) as a reference to measure the relative expression of the urease gene. The
results are shown graphically as copies of target mRNA per copies of a reference gene per
1000, based on the comparative CT method (5).
Data were expressed as mean  standard error of the mean (sem) of the biological samples.
Statistical analyses between groups were tested by the REST 2008 program (Relative
Expression Software Tool v 2.0.7, Corbett Research), that makes no assumptions about the
distributions, evaluating the significance of the derived results by Pair-Wise Fixed
Reallocation Randomization Test tool in REST (6).
cDNA synthesis for RT-qPCR
For cDNA synthesis, 400 ng of RNA were used. Prior to cDNA synthesis, samples were
treated for 30 min at 30 ºC with one U of DNaseI (Promega). The transcriptor First Strand
cDNA Synthesis Kit (Roche) was selected for cDNA synthesis. In this case, random
hexamers were used as a priming method, allowing cDNA synthesis from bacterial mRNA.
The whole procedure was performed according to the manufacturer's instructions, but an
additional denaturation step (10 min at 65 ºC) was applied to the RNA-random hexamers
mixtures to denature mRNA secondary structures that may impair hexamers binding to the
template.
RT-qPCR
All reactions were carried out in a Lightcycler 2.0 (Roche) with the kit FluoCycle IITM
SYBR® (Euroclone). Each PCR was run with 1 U of Taq polymerase and 250 mM of each
primer (see primer sequences in Table S2). Temperature profile was 95 ºC for 10 min
followed by 40 cycles of 95 ºC for 5 s, 55 ºC for 8 s, and 72 ºC for 20 s. Finally a melting
curve analysis was performed to ensure that only one product was present.
Supplementary Tables
Table S1. Protein content and composition of the cockroach diets.
Protein content
Composition
Dextrin
Low protein
High protein
Control
0%
5%
50%
~25%
4g
WSMa
4g
WSMa
4g
WSMa
96g dextrin
10g YEb
10g YEb
20g cellulose
106g cellulose
45g casein
Commercial dog food
41g dextrin
a
Wesson salt mixture (7).
Yeast Extract (Scharlau).
b
Table S2. Genes and primers used in dietary protein level experiments.
Gene
Primer
Sequence
Actin
95
96
512
URTOf
URTOr
ALNf
ALNr
ALTf
ALTr
BgGSaf
BgGSar
TU1f
TU1r
UC1f
UC1r
5’-TCGTTCGTGACATCAAGGAGAAGC-3’
5’-TGTCGGCAATTCCAGGGTACATGGT-3’
5’-AGCTTCCTGATCGTCAGGTGA-3’
5’-AAATCATTGGTGGGCTTCGTGGTC-3’
5’-TCACGTCTGGCAACGTTCTGTACT-3’
5’-TCTGACAGCAGAAACCTGTCACCA-3’
5’-GCTGCCCAAAGACGTTCCTTGTTT-3’
5’-GGAATTATGCACCTCGCTTCTCTC-3’
5’-CACTCCCTATTCTACTGTTCCGAC-3’
5’-TACAAAGATCCATTCAGGCCA-3’
5’-CACGTATGCCTTTGATTTGTGG-3’
5’-AAGGAAGAAGGAGGACGACACACT-3’
5’-TAGGCTGATGCAATTCCACCTCCA-3’
5’-GTCCAGCAACTGGAACTATAGCCA-3’
5’-CCTCCTGCACCTGCTTCTATTTGT-3’
Urate oxidase
Allantoinase
Allantoicase
Glutamine synthetase
Elongation factor EF-Tu
Urease
Table S3. Accession numbers in European Nucleotide Archive. The E-value and the species for the best hit of
each sequence after a Blastx against the NCBI is indicated.
Gene
Urate oxidase
Allantoinase
Allantoicase
Aspartate aminotransferase
Mitochondrial like
Cytoplasmic
Asparagine synthetase
Glutamate dehydrogenase, mitochondrial
Glutamine synthetase 2
Glutamate-semialdehyde dehydrogenase
Ornithine-δ-transaminase
Pyrroline-5-carboxylate reductase
Isozyme P5CR
Isozyme P5CR2
Phophoglycerate dehydrogenase
Phosphoserine transaminase
Phosphoserine phosphatase
Serine hydroxymethyl transferase
Accession number
HG965798
HG965799
HG965800
E-value (Species)
6e-145 (Solenopsis invicta)
0.0 (Zootermopsis nevadensis)
2e-38 (Bombyx mori)
HG965785
HG965786
HG965787
HG965788
HG965789
HG965801
HG965792
0.0 (Zootermopsis nevadensis)
2e-160 (Zootermopsis nevadensis)
0.0 (Zootermopsis nevadensis)
0.0 (Zootermopsis nevadensis)
0.0 (Zootermopsis nevadensis)
8e-61 (Papilio xuthus)
0.0 (Musca domestica)
HG965795
HG965796
HG965790
HG965794
HG965793
HG965791
2e-88 (Zootermopsis nevadensis)
6e-44 (Tribolium castaneum)
2e-104 (Zootermopsis nevadensis)
0.0 (Zootermopsis nevadensis)
1e-54 (Locusta migratoria)
0.0 (Culex quinquefasciatus)
Supplementary References
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