Average nucleotide identity (ANI) percentage among
Abstract
The genome and transcriptome of the endosymbiotic flavobacterium Candidatus Walczuchella monophlebidarum revealed its role in the synthesis of essential amino acids for its host, the wax cochineal Llaveia axin axin. There were, however, missing genes in the endosymbiont for some biosynthetic pathways. Here, we characterized TME1, another cochineal symbiont that may metabolically complement Walczuchella. TME1 was ascribed to the gammaproteobacterial genus Sodalis on a phylogenomic basis using gene sequences from 143 proteins core genome sequences and the core average nucleotide identity (ANI) confirmed its position. Additionally, we describe Sodalis as a coherent genus. TME1 genome is around 3.4 Mb and has complete gene sequences for the biosynthesis of 10 essential amino acids, for polyamines, flagella, nitrate respiration, and detoxification among many others. Transcripts from ovaries and bacteriomes allowed the identification of differentially transcribed genes from the endosymbionts and host. Highly transcribed genes were identified in TME1 and transcripts involved in amino acid biosynthesis were found. We review here that cosymbionts that derived from different bacterial classes and genera seem to be advantageous for insects that have Flavobacteria as the primary endosymbionts.
Keywords
- endosymbionts
- scale insect
- Gammaproteobacteria
- Sodalis-like
- Alphaproteobacteria
- fungi
1. Introduction
All organisms are inhabited by microbes that exert different effects on their hosts. In insects, there are many examples of beneficial associations with symbiotic microbes that have been linked to the insect ecological success. Symbionts that are vertically transmitted from mother to offspring and with an intrinsic interdependence with the insect host are considered as primary endosymbionts and they have reduced genomes [1, 2]; they do not grow on standard laboratory media. In theory, endosymbionts evolved from gut bacteria [3] that are largely more complex and may be determined by the diet and the environment. Primary endosymbionts may reside inside insect cells called bacteriocytes that may be found in specialized host structures called bacteriomes. Bacteriomes may be equivalent to plant-root nodules considering that they are host structures harboring particular bacterial species with specific roles [4]. But even in plants, cosymbionts have been encountered; for example, the slow-growing actinobacteria
In insects, cosymbiosis is not uncommon and there are cases in which two or more bacterial symbionts are found in the bacteriome [7, 8]. Additionally, other microbes including fungi may be found in the hemolymph or in different insect tissues [9–11]. Fungal symbionts may be found as well in specialized insect structures known as mycangia [12] or inside insect cells called mycetocytes [13].
In insects, primary bacterial endosymbionts synthesize essential amino acids or vitamins for their hosts and reside intracellularly in bacteriomes. In some cases, complementation of metabolic pathways seems to occur among different insect symbionts [14–17]. Additionally, cosymbionts may have different roles, and some have been implicated in defense [18–21], tolerance to stress [22], resistance to high temperatures [23–25], to virus [26–28], or may manipulate sex differentiation [29]. There is an example in which a secondary endosymbiont substituted a lost primary
Scale insects (Hemiptera: Coccoidea) feed on plant sap, which is a nutritionally poor diet that lacks most of the essential amino acids. Therefore, these insects have built up symbiotic associations with bacteria that can synthesize them. Most of the scale insect families that have been analyzed, such as Monophlebidae, Coelostomidiidae, Orthezidae, Phenacoccinae from Pseudococcidae, Coccidae, Lecanodiaspididae, Diaspididae, and a clade of Eriococcidae, harbor flavobacteria as primary symbionts and enterobacteria as secondary symbionts. [37–39]. It has been reported that the families from scale insects Dactylopiidae, some Eriococcidae, and Pseudococcinae from Pseudococcidae harbor different endosymbionts, which could indicate that they lost their flavobacteria and enterobacteria and acquired other endosymbionts [39]. Flavobacteria seem to be very ancient symbionts, perhaps starting symbiosis before the divergence of scale insects [39] (150–250 mya [40]). Although it has been suggested that Flavobacteria have cospeciated only within Monophlebidae, Coelostomidiidae, Ortheziidae, and Diaspididae [38–41], and host switches seem to have occurred in the other families [39]. Otherwise, enterobacteria have undergone more evolutionary events (losses, duplications, and host switches). Some scale insects have enterobacteria closely related to
The flavobacteria endosymbiont
2. Materials and methods
DNA, sequencing, and assembly were performed from bacteriomes (Illumina HiSeq 2000) and from the homogenized of female adults (pyrosequencing) of
Comparative phylogenomic analysis was performed with 20 genomes of gammaproteobacteria from GeneBank. Gene calling of all genomes was performed using GeneMark version 2.5 [53]. The pangenome and core genome from orthologous genes of all strains were obtained by GET_HOMOLOGUES version 2.0 software [54] with -A -c -t 0 -M -n 35 and -A -c -t 0 -G -n 35 parameters. We selected a set of 143 unique single-copy orthologous genes from the core genome. Translated coding sequences of each gene were concatenated using BioEdit Version 7.2.5 and aligned with Clustal Omega version 1.2.1 [55]. Prottest3 version 3.4.2 [56] was used to select the best amino acid substitution model using the AICc correction. The edited alignment contained 47,803 amino acid positions. Maximum likelihood phylogeny was performed by PhyML software version 3.1 [57] using the CpREV model with the Shimodaira–Hasegawa-like procedure for internal branch support [58]. The genome of
Comparative genomics was carried out with the following
RNA was extracted from the bacteriome of
To determine the uric acid and uricase activity,
3. Results
We found gene sequences of an enterobacterium (gammaproteobacterium) related to
TME1 was compared with the ANI (average nucleotide identity) metric to other
Sodalis str. TME1 | Sodalis pierantonius SOPE | Sodalis praecaptivus HS1 | Sodalis glossinidius str. morsitans | Sodalis-like str. PSPU | Sodalis-like str. SPI-1 | |
---|---|---|---|---|---|---|
91.24 | 91.04 | 91.27 | ||||
89.81 | 89.67 | 89.87 | ||||
92.25 | 91.94 | 92.15 | 89.91 | 85.86 |
The draft assembly of the enterobacterial endosymbiont
We obtained 11,042,037 and 11,042,428 reads from the cDNA sequence of the bacteriome and the ovaries, respectively. These two organs were selected for studying the differentially expressed genes because endosymbionts are transferred from bacteriomes to the ovaries for vertical transmission to their offspring. It was expected to find genes related to the migration of the endosymbionts from the bacteriome and the colonization of the ovaries. Reads mapped to the reference genomes are shown in Table 2. The number of genes that were statistically differentially expressed is shown in Table 3.
Reference genome | Bacteriome | Ovaries |
---|---|---|
2,019,585 | 2,008,381 | |
3,082,319 | 2,912,207 | |
1,052,077 | 87,502 | |
409,128 | 483,601 |
Reference genome | Bacteriome | Ovaries |
---|---|---|
494 | 680 | |
244 | 280 | |
2 | 89 | |
66 | 50 |
Sodalis TME1 | Insect | ||
---|---|---|---|
Putative hydrolase | T3SS-secreted effector | Chaperon Hsp70 | |
Chaperones GroEL, GroES | Allantoinase | ABC transporters | |
Hypothetical proteins | Hypothetical proteins | Antiparasitic-like peptide | |
ATP synthase B subunit | NAD biosynthesis | Asparaginase | |
Amino acids biosynthesis genes | FtsE cell division gene | Unknown genes | |
Transcriptional regulation | Extracellular glutamate receptor channel | ||
Flagellum synthesis | Phospholipids synthesis | ||
Transcriptional regulation | |||
ATP synthase B and A subunits (pseudogenes) | Hypothetical proteins | ATPase subunit | |
AhpC oxidative stress gene | NAD biosynthesis | Transmembrane transporters of sugars and amino acids | |
Glycoprotease | Flagellum synthesis | Peptidoglycan-binding protein | |
Amino acids biosynthesis genes | FtsE cell division gene | Lysozyme | |
Cytochrome c oxydase | Glycolysis | Unknown genes | |
SecY translocase | Phage lysozyme | Transcriptional regulation | |
Hypothetical proteins | Transcriptional regulation | Phospholipids synthesis |
From the ovary tissue, we found differential expression of
In the bacteriome, the enterobacterium TME1 showed very strong overexpression of a gene that codes an effector protein possibly secreted by the type III secretion system (TTSS), expressed 66.8-fold compared to its expression in the ovaries. Also, a gene that codes an allantoinase that participates in uric acid metabolism is highly overexpressed in the bacteriome, showing a 50-fold change. Other genes with overexpression in the bacteriome are four ABC transporters, a peroxidase, the heme synthase, two genes related to nucleotides biosynthesis, two genes related to lipid A biosynthesis, and two genes of the type III secretion system (Table 4).
In the ovary, TME1-overexpressed genes were related to NAD synthesis, carbohydrate metabolism, stress response, and some transporters and transcriptional regulators (Table 4).
Among the insect differentially expressed genes in the bacteriome there were 19 putative transporters (for amino acids, carbohydrates, vitamins, drugs, or unknown substrates), five genes related to defense systems including an antiparasitic peptide with identity to Drosomycin, three from
On the other hand, we found that in the insect, in the ovaries there was overexpression of 15 transporters, 17 immune response genes, some genes related to heat shock, desiccation, oxidative stress, and hypoxia response, and genes related to lipids, vitamins, carbohydrates, nucleotides, amino acids, and chitin synthesis and metabolism (Table 4).
4. Discussion
Due to the annual cycle of the wax cochineal, we are only able to collect insects once a year during the rainy season. It is worth mentioning that in 2015 and 2016, we did not find cochineals in many of the places where we had collected previously. Considering the menace of mosquitoes transmitting Zika, or Chikungunya, extensive fumigations with chemical insecticides have been carried out in many places in Mexico, especially in Chiapas. The relation to the diminished populations of cochineals remains to be established.
A previous survey of symbiotic bacteria from scale insects in Mexico revealed the prevalence of Flavobacteria and Gammaproteobacteria [39]. Some of the Gammaproteobacteria had 16S ribosomal gene sequences closely related to those of TME1, and thus they may be considered as
While Flavobacteria and insects showed a co-divergent pattern of evolution, the phylogenetic relationships of the Gammaproteobacteria and insects were not parallel, indicating multiple enterobacterial transfers among the different hosts, and a more recent and less dependent symbiosis. In agreement, the genome size of the gammaproteobacterium TME1 is much larger than that from the primary endosymbiont from wax cochineals, the Flavobacteria
The genome from the gammaproteobacterium TME1 (3.4 Mb) is within the range of those from other
TME1 has biosynthetic pathways for all essential amino acids and may supply the needs of the wax cochineal and of
In
Uric acid and uricase activity were detected in
By reverse transcriptase-polymerase chain reaction (RT-PCR) using primers targeted to
Here, we used the term symbiome [27] to refer to the group of primary and secondary (cosymbionts) endosymbionts (and/or their genomes), residing in a host. We consider that the term symbiome is more adequate than the terms endosymbiotic community or consortium that are sometimes used instead.
The cosymbionts of different Flavobacteria in scale insects are diverse lineages of related Gammaproteobacteria [39]. Similarly, the cosymbionts of
Acknowledgments
AVPL is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and received the 331625 fellowship from Consejo Nacional de Ciencia y Tecnología (CONACyT).
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