Supplemental figure 1: Heat map showing enrichment of
assembled contigs according to the 4 different libraries used in this work.

Supplemental figure 2: Phylogenetic analysis of the salivary
antigen 5 family of Haematobia irritans
and their close relatives of the Diptera family. The evolutionary history was
inferred by using the Maximum Likelihood method based on the JTT matrix-based
model (Jones et al., 1992). The bootstrap consensus tree inferred from 500
replicates (Felsenstein, 1985) is taken to represent the evolutionary history
of the taxa analyzed. Branches corresponding to partitions reproduced in less
than 50% bootstrap replicates are collapsed. Initial tree(s) for the heuristic
search were obtained by applying the Neighbor-Joining method to a matrix of
pairwise distances estimated using a JTT model. A discrete Gamma distribution
was used to model evolutionary rate differences among sites (5 categories (+G,
parameter = 2.0103)). The analysis involved 66 amino acid sequences. All
positions with less than 50% site coverage were eliminated. That is, fewer than
50% alignment gaps, missing data, and ambiguous bases were allowed at any
position. There were a total of 257 positions in the
final dataset. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013).  The
sequence names start with the first three letters of their genus name, followed
by the first three letters of the species name, followed by their GenBank
accession. The H. irritans sequences
have their expression index following their GenBank accession. The sequences
from H. irritans are marked with a
red circle, those of Stomoxys calcitrans
with a blue circle, and that from Dipetalogaster
maxima is marked with a green circle.

Supplemental figure 3: Phylogenetic analysis of the
Hematobin family of Haematobia irritans
and their close relatives of the Diptera family. The evolutionary history was
inferred by using the Maximum Likelihood method based on the Whelan And Goldman
model (Whelan and Goldman, 2001). The bootstrap consensus tree
inferred from 500 replicates (Felsenstein, 1985) is taken to represent the evolutionary history
of the taxa analyzed. Branches corresponding to partitions reproduced in less
than 50% bootstrap replicates are collapsed. Initial tree(s) for the heuristic
search were obtained by applying the Neighbor-Joining method to a matrix of
pairwise distances estimated using a JTT model. A discrete Gamma distribution
was used to model evolutionary rate differences among sites (5 categories (+G,
parameter = 6.4363)). The analysis involved 30 amino acid sequences. All
positions with less than 50% site coverage were eliminated. That is, fewer than
50% alignment gaps, missing data, and ambiguous bases were allowed at any
position. There were a total of 158 positions in the
final dataset. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). The hematobin
sequence showing macrophage inhibition activity (Breijo et al., 2018)
is marked with a red circle. The names of the sequences originating in this
work start with the letters “ab” or “tr”. The
remaining sequences are named with their GenBank accession followed by the
first 3 letters of their genus name followed by the first three letters of
their species name.

Supplemental figure 4: Structural highlights and phylogenetic insights of
Haematobia irritans Nora virus (HiNV). (A) Genome graphs depicting architecture
and predicted gene products of HiNV. The coding
sequences (CDS) are shown in light orange arrow rectangles, start and end
coordinates are indicated. Gene products are depicted in curved yellow
rectangles and size in aa and weight in kDa is indicated below. Predicted
domains using the NCBI-CDS or HHPred best-hit regions
are shown in curved green or orange rectangles, respectively. Abbreviations:
VSR, viral silencing suppressor; HEL, helicase domain; PRO, serine protease
domain; RdRP, RNA dependent RNA polymerase; TM,
trans-membrane domain; OCP, outer capsid protein sigma-1 domain; VP4-A/C, Nora
virus coat protein subunit structural domains . (B) HiNV virus
RNA levels normalized as reads per million (RPM) of midgut samples SRR5136555
and SRR5136554 (MG), and salivary gland samples SRR5136552 and SRR5136553 (SG).
(C) Sequence similarity comparison
of the RNA genomes (% nt, upper panel) and predicted gene products (% aa, lower
panel) of HiNV-URU sequence with the reference
sequence of Nora virus (GenBank NC_007919). (D) Maximum likelihood phylogenetic tree based on amino acid
alignments of the capsid protein of HiNV and reported
Nora viruses. The tree is rooted at Nasonia vitripennis virus, host linked clades are indicated by
colored rectangles, and representative hosts are depicted as silhouettes. In
the lower left corner a radial unrooted tree is
depicted showing flies and bee associated Nora viruses.  The scale bar indicates the number of
substitutions per site. Node labels indicate FastTree
support values. The viruses used to construct the tree, and their accession
numbers are Drosophila simulans Nora virus
(AKH67661), Apis mellifera Nora virus (ARO50052), Drosophila suzukii Nora virus (AWA82260), Drosophila melanogaster Nora
virus (YP_004849310), Drosophila subobscura Nora
virus (YP_009047188), Drosophila immigrans Nora virus
(YP_009047192), Nasonia vitripennis
virus (ACN94448), Spodoptera exigua
virus AKJ-2014 (AHX00966), Helicoverpa armigera Nora virus (AZF98739). (E) Highly conserved C-region of MAFTT alignment of capsid proteins
of Nora viruses. Similarity is shown from white to black aa shading, and
numbering corresponds to consensus alignment. (F)  Representative RNA
mapping results of H. irritans midgut
sample SRR5136554 to the VP4 CDS terminal region of HiNV-URU
sequence showing consistent support of the premature translation stop signal of
the capsid protein.

Supplemental
figure 5: Structural
highlights and phylogenetic insights of Haematobia irritans densovirus
(HiDV). (A)
Genome graphs depicting architecture and predicted gene products of HiDV. The coding sequences (CDS) are shown in light orange
arrow rectangles, start and end coordinates are indicated. Gene products are
depicted in curved yellow rectangles and size in aa and weight in kDa is
indicated below. Predicted domains using the NCBI-CDS are shown in curved green
rectangles. Abbreviations: HP1-2, hypothetical protein 1-2; Parvo_NS1,
parvovirus non-structural protein NS1 domain; Parvo_CP,
Parvovirus coat protein N domain; Denso_VP4, Capsid protein VP4 domain. (B) Radial unrooted maximum likelihood
phylogenetic tree based on amino acid alignments of the capsid protein of HiNV and reference sequences of members of the Parvoviridae
family of viruses. Clusters of genera are collapsed at the nodes. Sub-family Densovirinae and Parvovirinae are
indicated by light blue and light red colors, respectively. Genera at the node
are abbreviated with the first three letters of the genus. (C) HiDV virus
RNA levels normalized as reads per million (RPM) of midgut samples SRR5136555
and SRR5136554 (MG), and salivary gland samples SRR5136552 and SRR5136553 (SG).
(D) Representative RNA mapping
results of H. irritans sample
SRX3636749 (USA) to the HiDV-URU sequence showing
consistent variable sites depicted as green bars. The lower panel shows a
magnification of the mapping results of a mid region
of the HiDV VP CDS where variable sites are indicated
by colored bases, highlighting mismatches with the HiDV-URU
sequence. Specific variants are numbered based on Table 8. (E) Maximum
likelihood phylogenetic tree based on amino acid alignments of the VP protein
of HiDV and proposed and ICTV accepted members of
subfamily Densovirinae.
The tree is rooted at genus Hepandensovirus, genera are indicated by colored rectangles,
and representative hosts are depicted as silhouettes. The scale bar indicates
the number of substitutions per site. Node labels indicate FastTree
support values. The viruses used to construct the tree, and their accession
numbers are available in Table 9.

Breijo, M., Esteves, E., Bizzarro, B.,
Lara, P.G., Assis, J.B., Rocha, S., Pastro, L., Fernandez, C., Meikle, A.,
Sa-Nunes, A., 2018. Hematobin is a novel immunomodulatory protein from the
saliva of the horn fly Haematobia
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