TEXT S11: THE 9 DESATURASE GENES
Elizabeth Cash, Martin Helmkampf, and Jürgen Gadau
School of Life Sciences, Arizona State University, Tempe, AZ 85287, United States of America
Among the key features distinguishing social insects from other, non-social
animals is the governance of colonies belonging to the former by complex
communication systems. While the presence of these systems may be evident in such
phenomena as colony recognition and task differentiation [1,2], the intricacies of such
communications remain only limitedly defined. Even so, promising developments in the
study of cuticular hydrocarbons (CHCs) suggest that such molecules might serve a
critical, if not definitive, role in social insect nestmate recognition [3,4]. However, several
studies in two leaf-cutter ant species, Atta laevigata and Atta cephalotes, show that
recognition cues can originate from sources other than through the cuticle, namely from
the mandibular glands and abdominal exocrine glands [5,6]. These findings suggest that
colony recognition among social insects, such as Atta cephalotes, results from an
intercourse of multiple variables, including: CHC profiles, gland components, and/or
environmentally derived compounds [7,8].
The best studied among colony recognition signals are the CHCs, of which
nearly 1,000 compounds have been described among 78 species investigated [9]. From
these, two biochemical pathways are largely shown to alter n-alkanes – the
incorporation of double bonds and the addition of methyl braches. These two
compound-groups may factor substantially into colony recognition given their more
discernible and variable nature relative to linear alkanes [9]. Although little is known
about CHC related genes in social insects, studies of CHC components in Drosophila
melanogaster show that desaturases, elongases, and carboxylases each contribute to
CHC biosynthesis [10]. Well studied among these are three Drosophila desaturase
genes, desat1, desat2, and desatF, which insert carbon-carbon double bonds into nalkanes forming monoenes and dienes, and contribute to Drosophila melanogaster
CHC alkene synthesis and CHC phenotypic variation [11,12]. Atta cephalotes workers,
notably, have several CHC alkenes [13], therefore making these three desaturase
genes, Drosophila melanogaster desat1, desat2, and desatF, strong candidates for
identifying Atta cephalotes genes involved in CHC alkene biosynthesis.
Gene Annotation and BLAST Analysis
We performed manual annotation by using the protein amino acid sequences of
the three Drosophila melanogaster desaturase genes (desat1, desat2, and desatF)
obtained from the NCBI web accessible database. These three query sequences were
then separately analyzed with the BLAST package program [14] to search for similar
amino acid sequences (significant alignment e-value threshold = e–10) in the Atta
cephalotes genome. Results from our analysis of the Atta cephalotes genome show
eleven complete ∆9 desaturase-like amino acid sequences (two with EST evidence),
and two fragmentary ∆9 desaturase-like amino acid sequences (one with EST
evidence). Interestingly, nine of the complete sequences and one of the fragments were
grouped on a single scaffold in Atta cephalotes, with the nine complete sequences
being found together along a relatively small 200 kb region of the genome, and
markedly differs in comparison to a more scattered arrangement of the seven ∆9
desaturase genes found on chromosome three in Drosophila melanogaster (Fig. 1). We
determined homology relationships with Drosophila melanogaster desaturase genes via
reciprocal BLAST of the Atta cephalotes desaturase genes against the Drosophila
melanogaster genome in NCBI, and found that six of the complete gene sequences and
the two fragment sequences in Atta cephalotes were best aligned with the Drosophila
melanogaster ∆11 desaturase gene CG9747, three of the complete sequences were
most similar to Drosophila melanogaster ∆9 desaturase gene desat1, one of the
complete sequences aligned best with Drosophila melanogaster ∆9 desaturase gene
CG9743, and the last of the complete sequences was best matched with Drosophila
melanogaster ∆9 desaturase gene CG15531. Notably, none of the sequences were
most similar to Drosophila melanogaster ∆9 desaturase genes desat2, desatF, or
CG8630.
Phylogenetic Analysis
Using the ∆9 desaturase genes found in Atta cephalotes and six other insect taxa
with completed genomes, we performed a phylogenetic analysis to identify the
relationships between the desaturase genes of Atta cephalotes and other insects (two
Apis mellifera partial gene sequences were not included, however, in order to improve
the final length of the trimmed dataset: one similar to CG15531, and one similar to
CG8630). The resulting amino acid sequences of 74 homologous genes were aligned
using the L-INS-i algorithm implemented in MAFFT v6 [15]. Poorly aligned amino acids
were eliminated by Aliscore v1 [16] set with default parameters, giving a final dataset
composed of 293 amino acid positions. We used ProtTest [17] to determine the
evolutionary model with the best fit to this dataset according to the Akaike Information
Criterion corrected for small sample size, and found this to be the LG+G model. A
maximum likelihood tree was then reconstructed using RAxML v7.2.6 [18], and nodal
support values were obtained by rapid bootstrap analysis with 500 replicates.
Our phylogenetic analysis resulted in a maximum likelihood tree revealing five
major clades within the ∆9 desaturase gene family, of which, Atta cephalotes was
represented in four (Fig. 2). Two clades, clade E and clade D, are very well supported
(respectively, BS = 100 and BS = 98) and consist of single copy genes of most of the
represented taxa, including Atta cephalotes. All of the genes in clade E (which had
previously been assigned names based on reciprocal BLAST results to Drosophila
melanogaster) were found to cluster with other ∆9 desaturase CG15531 genes, and
both reciprocal BLAST results and phylogenetic results suggest that this gene was lost
in the red flour beetle, Tribolium castaneum. Clade D, similarly, is comprised of ∆9
desaturase CG9743 genes that are supported by both reciprocal BLAST and
phylogenetic analyses (with the exception of a Nasonia vitripennis gene, Nvit_desatC_c,
found to be most similar to CG9743 according to reciprocal BLAST, but which groups
with a separate clade, clade C, in phylogenetic analyses, see below). The results for
clade D additionally suggest losses in Acyrthosiphon pisum and Nasonia vitripennis.
Remarkably, the contradiction between sequence similarity (based on reciprocal
BLAST) and phylogeny in Nvit_desatC_c exists for all genes present in the moderately
supported (BS = 75) clade C due to the absence of a Drosophila melanogaster gene in
this clade, and highlights the inaccuracy in solely using reciprocal BLAST analyses for
determining homology relations. Clade C is also noteworthy for a small, strongly
supported (BS = 100) expansion in Nasonia vitripennis, but has evidently been lost in
the aculeate lineages, Atta cephalotes and Apis mellifera. A fourth well supported (BS =
95) monophyletic group of the desaturase CG9747 (clade B) is distinguished by several
gene expansions in Atta cephalotes and Nasonia vitripennis, which seem to have
occurred both before and after the split of these lineages, yet is surprisingly not present
in Apis mellifera. The remaining 9 desaturases are found in clade A, a large and
weakly supported (BS < 50) group with minimal internal resolution, which contains
numerous genes in all represented taxa. This group includes the original set of
Drosophila melanogaster query genes, desat1, desat2 and desatF, which appear to
have arisen from dipteran or Drosophila specific gene duplications, although the timing
of these events is ambiguous relative to the split between the lineages leading to
Drosophila and Anopheles. A fourth Drosophila melanogaster gene, CG8630, also falls
into this clade, however, due to contradictory reciprocal BLAST results its orthologs
cannot be identified reliably (with the exception of two A. pisum genes and one
Tribolium castaneum gene). Given this, all other genes that are phylogenetically more
closely related to CG8630 than to desat1 are identified orthologous to the latter
according to the best reciprocal BLAST results, which is the case for to two Atta
cephalotes genes, Acep_CG8630_a and Acep_CG8630_b. Finally, the third Atta
cephalotes gene in clade A, Acep_desat1, can be recognized as a true ortholog of the
Drosophila melanogaster desat1 according to both the phylogenetic and reciprocal
BLAST analysis, and therefore makes a leading candidate gene for further study of
CHC alkene biosynthesis in Atta cephalotes.
References
1. Hölldobler B, Wilson EO (1990) The ants. Cambridge, Mass.: Harvard University
Press. xii + 732 p.
2. Payne C, Tillberg C, Suarez A (2004) Recognition systems and biological invasions.
Annales Zoologici Fennici 43: 843-858.
3. Lahav S, Soroker V, Hefetz A, Vander Meer RK (1999) Direct Behavioral Evidence
for Hydrocarbons as Ant Recognition Discriminators. Naturwissenschaften 86:
246.
4. Wagner D, Tissot M, Cuevas W, Gordon DM (2000) Harvester Ants Utilize Cuticular
Hydrocarbons in Nestmate Recognition. Journal of Chemical Ecology 26: 2245.
5. Jaffe K (1983) Chemical communication systems in the ant Atta cephalotes. In:
Jaisson P, editor. Social Insects in the Tropics. Paris: University of Paris, Nord.
pp. 165-180.
6. Hernández JV, Goitía W, Osio A, Cabrera A, Lopez H, et al. (2006) Leaf-cutter ant
species (Hymenoptera: Atta) differ in the types of cues used to differentiate
between self and others. Animal Behaviour 71: 945.
7. Hefetz A, Errard C, Chambris A, Negrate A (1996) Postpharyngeal gland secretion as
a modifier of aggressive behavior in the myrmicine ant Manica rubida. Journal of
Insect Behavior 9: 709.
8. Carlin NF, Hölldobler B (1987) The Kin Recognition System of Carpenter Ants
(Camponotus spp.): II. Larger Colonies. Behavioral Ecology and Sociobiology 20:
209.
9. Martin S, Drijfhout F (2009) A Review of Ant Cuticular Hydrocarbons. Journal of
Chemical Ecology 35: 1151.
10. Gleason JM, Jallon J-M, Rouault J-D, Ritchie MG (2005) Quantitative Trait Loci for
Cuticular Hydrocarbons Associated With Sexual Isolation Between Drosophila
simulans and D. sechellia. Genetics 171: 1789.
11. Dallerac R, Labeur C, Jallon J-M, Knipple DC, Roelofs WL, et al. (2000) A Delta9
desaturase gene with a different substrate specificity is responsible for the
cuticular diene hydrocarbon polymorphism in Drosophila melanogaster.
Proceedings of the National Academy of Sciences of the United States of
America 97: 9449.
12. Legendre A, Miao X-X, Da Lage J-L, Wicker-Thomas C (2008) Evolution of a
desaturase involved in female pheromonal cuticular hydrocarbon biosynthesis
and courtship behavior in Drosophila. Insect Biochemistry and Molecular Biology
38: 244.
13. Richard F-J, Poulsen M, Drijfhout F, Jones G, Boomsma J (2007) Specificity in
Chemical Profiles of Workers, Brood and Mutualistic Fungi in Atta, Acromyrmex,
and Sericomyrmex Fungus-growing Ants. Journal of Chemical Ecology 33: 2281.
14. Korf I, Yandell M, Bedell J (2003) BLAST. Sebastopol, CA: O'Reilly & Associates.
15. Katoh K, Misawa K, Kuma Ki, Miyata T (2002) MAFFT: a novel method for rapid
multiple sequence alignment based on fast Fourier transform. Nucleic Acids
Research 30: 3059.
16. Kuck P, Meusemann K, Dambach J, Thormann B, von Reumont B, et al. (2010)
Parametric and non-parametric masking of randomness in sequence alignments
can be improved and leads to better resolved trees. Frontiers in Zoology 7: 10.
17. Abascal F, Zardoya R, Posada D (2005) ProtTest: selection of best-fit models of
protein evolution. Bioinformatics 21: 2104.
18. Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic
analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688.
Figure 1. Arrangement of the eleven complete ∆9 desaturase genes and two fragmentary ∆9 desaturase
genes found in the Atta cephalotes genome, in comparison to the seven ∆9 desaturase genes found on
chromosome three in the Drosophila melanogaster genome.
Figure 2. Unrooted maximum likelihood tree of all ∆9 desaturase genes found in the genomes of Atta
cephalotes (Acep, in bold), Nasonia vitripennis (Nvit), Apis mellifera (Amel), Acyrthosiphon pisum (Acpi),
Tribolium castaneum (Tcas), Drosophila melanogaster (Dmel), and Anopheles gambiae (Agam). Gene
labels reflect phylogenetic relationships to D. melanogaster ∆9 desaturase genes, excpect in clade C
where D. melanogaster genes are not found, and are thus newly named “desatC”. Support values > 50
based on 500 rapid bootstrap replicates are shown at the nodes of the tree. Asterisks indicate Atta
cephalotes genes closely linked together (see Figure 1).