Archaea, the unseen kingdom in the gut microbiome of Anastrepha obliqua

The fruit y Anastrepha obliqua is an economically important pest. The sterile insect technique is used to control this pest; it involves mass production and release of sterile ies to reduce the reproduction of the wild population. As noted in different tephritidae, the performance of sterile males may be affected by the assimilation of nutrients under mass-rearing conditions. In the wild, the y’s life cycle suggests the acquisition of different organisms that could modulate its tness and physiology. For A. obliqua, there is no information regarding microorganisms other than bacteria. This study analyzed bacteria, fungi, and archaea communities in the A. obliqua gut through denaturing gradient gel electrophoresis (DGGE) proles of 16S (using different set of primers for bacteria and archaea) and 18S ribosomal DNA markers. We found that wild ies presented higher microbial diversity than laboratory samples related to fructose assimilation. In contrast, laboratory microbial species suggest that microorganisms have led to a specialized metabolism to process nutrients associated to an articial diet. We showed that by employing different primer sets aimed at the same molecular marker but targeting diverse hypervariable regions of bacteria and archaea 16S rDNA, it was possible to identify species that have not been previously described in this fruit y. Here, the archaea kingdom is suggested as an important player in y metabolism. This report of the intestinal microbial (bacteria, archaea, and fungi) composition of A. obliqua, contributes to our understanding of the role of microorganisms in the development and physiology of the ies.


Introduction
Fruit ies (Diptera: Tephritidae) encompass ~ 70 species, which infect more than 30 fruit species, leading to worldwide economic impacts (Qin et al. 2015). In Mexico, despite being cosmopolitan, the Mediterranean fruit y Ceratitis capitata has been controlled by the sterile insect technique (SIT). However, the fruit y Anastrepha obliqua (Macquart) is a pest that causes losses of fruit crops; from January to July 2018 there were losses of 22 million tons of mango (Mangifera indica) and Spondias (Spondias purpurea and S. mombin) fruits (SENASICA 2018). SIT is a biological technique without adverse impact on biodiversity and the environment, that involves the systematic mass release of irradiated sterile adult competitive and ying males, which induce sterility in the wild population, by preventing offspring (KNIPLING 1959; Montoya and Toledo 2010).
Different efforts had been made to optimize the e ciency of SIT by enhancing the quality of sterile insects (Ami et al. 2010). In this sense, nutrient assimilation is a critical factor for the quality of the released sterile insects. Composition of the arti cial diet, enzymatic metabolic machinery, and microorganisms harbored in the y's gut modulate assimilation, contributing to y tness (Rivera-Ciprian et al. 2017; Rempoulakis et al. 2018).
Additionally, the wildlife cycle of the fruit y suggests the acquisition of different microorganisms, which exploit nutrients from the natural diet, modulating its biology (Ben-Yosef et al. 2014). Therefore, the y's gut ecology must be determinative in the modulation of y tness.
The microorganisms present within a host, known as microbiota, are generally well known for modulating host health and tness (Thaiss et al. 2016).
In fruit ies, most of the microbial diversity studies have focused on elucidating the role of bacteria housed in the gut of different y genera, such as Ceratitis, Drosophila, Bactrocera, Helaeomyia, and Anastrepha (Kuzina et  For the related y, Anastrepha ludens, bacteria such as Citrobacter, Enterobacter, Klebsiella, Providencia, and Pseudomonas have been found in its intestinal tract (Kuzina et al. 2001). To date, studies on Anastrepha spp. have mainly focused on bacteria, and have not investigated other associated microorganisms, which could also modulate physiology and tness. Thus, knowing the ecology of the y intestine would provide a better understanding of microbiota-insect interactions, multi-species community structure, and its effect on the host. To determine the microbiota diversity associated with the gut of A. obliqua, we employed 16S and 18S PCR-denaturing gradient gel electrophoresis (DGGE) to describe the diversity of fungi, bacteria, and archaea present in the intestinal tract of wild and laboratory ies. Moreover, we used cloning techniques of 16S-ribosomal DNA (rDNA) coupled with phylogenetic analyses to gain a better insight into the species harbored in the y intestine. This knowledge will provide the basis for further studies of the microbiota in the digestive tract of A. obliqua, which could improve the success of sterile insect release.

Biological material
The larvae and adult A. obliqua ies were obtained from a mass-rearing colony at Moscafrut Facility in Metapa de Domínguez, Chiapas, Mexico. Third instar larvae were harvested and fed a diet containing 19% corn cob powder (Mafornu, Cd. Guzmán, Jalisco, Mexico), 5 adult specimens were collected after emerging from the pupae stage on vermiculite incubated at approximately 14 days and fed with hydrolyzed protein:sugar (in a 1:3 ratio), and water until collection at 12 days of age. Wild larvae were obtained from infested mango fruits provided by the Junta Local de Sanidad Vegetal del Soconusco, Chiapas. Third instars were obtained from infested mango fruits collected in the surroundings of the town of Tuxtla Chico, Chiapas, Mexico. Wild adult ies were caught during the harvest in mango orchards in the same town using modi ed MacPhail traps baited with hydrolyzed protein as an attractant to catch live adults (Enkerlin 2018). Flies were taxonomically identi ed using the keys previously described (FAO/OIEA 2018). All samples were washed twice with 70% ethanol. Dissections were performed to extract the digestive tract under sterile conditions in a laminar ow hood and stored in 70% ethanol at -20°C until processing.

DNA extraction
Before extraction, samples (0.3 g) of the midgut were washed twice with 700 µl of 20 mM EDTA. DNA was extracted following the phenol-chloroform protocol (Sambrook 2001) with some modi cations. Brie y, 500 µl of 2 mM EDTA pH 8, 500 µl phenol pH 8.0, 0.25 g of glass beads, and 60 µl 20% SDS were added to macerated midguts and homogenized for 4 min. DNA puri cation was performed with chloroform and centrifuged at 13,000 x g. Then, 2.7 M sodium acetate and two equal volumes of absolute cold ethanol were added to the aqueous phase, which was incubated for 40 min at -70°C to precipitate DNA, centrifuged 35 min at 16,000 x g, washed with 70% ethanol, decanted, and allowed to dry at 37°C. The DNA pellet was resuspended in 50 µl water with 1.5 µg/µl RNAse and incubated for 1 h at 37°C. The obtained genomic DNA was puri ed using the Soil Microbe DNA kit MiniPrep ZR ™ (Zymo Research, USA) according to the manufacturer's instructions. Each sample was analyzed by 0.8% agarose gel electrophoresis to verify quality and purity.
DNA extraction from legs was performed using a single laboratory adult specimen that was frozen at -20ºC. The legs were dissected, placed in a 1.5 ml tube, and washed with 70% ethanol ve times. DNA extraction was performed as described above.

PCR-DGGE analysis
Ampli cation of molecular markers was performed using the primers listed in Table 1, as follows. Bacterial 16S rDNA fragments were performed with primers F984GC and R1378 (Nübel et al. 1996). Ampli cation of Gram-positive bacteria (Actinomycetes) 16S rDNA was performed with a nested PCR employing the primer F243 and R1378 for the rst reaction to Gram positive population enrichment, and F984GC and R1378 in the second (Heuer et al. 1997). Archaea identi cation was performed using a nested ampli cation using primers 21F and 958R (DeLong 1992) in the rst PCR ampli cation; Parch519F and Arch915R (Coolen et al. 2004). To verify the presence of fungi, amplicons were obtained with primers NS1 (White et al. 1990) and GCfung (May et al. 2001). The primers alignment and PCR conditions are found at Online Resource 1. The DGGE band pro les indicate the different microbial communities from the y midgut (Online Resource 2). The pro les were compared using the Jaccard index (J), which allows analysis of the biodiversity found in a sample where the maximum value indicates a greater diversity. This was calculated according to the following formula: J = nAB (nA + nB -nAB) -1, where nAB is the number of bands in common between lanes A and B, nA is the total number of bands in lane A, and nB is the total number of bands in lane B.
The Shannon index (H') allows comparisons of community similarity between two samples, where a value of 1.0 (or 100%) corresponds to communities that share an identical pattern, and a value of 0 indicates that no difference exists. This index was calculated using the following equation: H' = -(ni/N) (log ni/N), where H' is the diversity, and ni/N is the number of individuals of the species given by band intensity (ni) to the total subjects (N, the total intensity of all bands of the same sample); i.e., the relative abundance of species. Band intensity re ects the abundance of the same (Eichner et al. 1999) and theoretically represents a genus, in this case, analyzed by Molecular Kodak Imaging software.

PCR-DGGE analysis
The 16S rDNA amplicons for bacteria, and archaea were obtained as described above. The puri ed PCR products were cloned into a p-JET vector with CloneJet PCR Cloning Kit (Thermo Scienti c, Waltham, MA, USA) and transformed into E. coli DH5-α cells (Sambrook 2001). Clones were sequenced using pJET primers by capillary sequencing at Macrogen Inc. (Korea). The sequence chromatograms were visually inspected and manually trimmed using AliView software (Larsson 2014). To identify closely related 16S rRNA genes, the remaining sequences were analyzed with the BlastN tool at 99% identity against the 16S archaea and bacteria database at NCBI. Those sequences were submitted to NCBI under the accession numbers shown at Table 2. Multiple sequence alignment was performed using ClustalW (Thompson et al. 1994). And the best suitable model was employed to construct the phylogenetic tree using Molecular Evolutionary Genetics Analysis (MEGA6) (Kimura 1980;Tamura et al. 2013). The accuracy of the tree topology was performed by 1000 bootstrap replicates (Felsenstein 1985).
Sample codes correspond at the label assigned to our samples; NCBI number is the assigned number after submission. LL-Laboratory larvae (mass-reared), LA-Laboratory Adult (mass-reared); WL-Wild larvae, WA-Wild adult.
The 16S rDNA clones of bacteria and archaea communities obtained from wild and laboratory adult and larvae ies were loaded into Geneious software version 11.1 (Kearse et al. 2012). Subsequently, all submitted NCBI sequences as well as those which display robust electropherogram (Online Resource 3) were manually curated and merged to gain a general view of the global biological diversity harbored at A. obliqua. Clean sequences were then analyzed using the 16S diversity tool in Geneious software.

Results And Discussion
Bacteria communities In related fruit-y pests such as Anastrepha obliqua, A. ludens, and Ceratitis capitata both, their gut enzymatic activity, and the presence of grampositive and gram-negative bacteria modulate the food bio-assimilation capability leading effects on host tness (Rivera-Ciprian et al. 2017; Kuzina et al. 2001;Ami et al. 2010; Gallo-Franco and Toro-Perea 2020). Because of this, we got interested in disclosing A. obliqua's bacterial microbiota diversity. For this, DNA was extracted from the digestive tract of wild adult and larvae (WA and WL, respectively) and laboratory (LA and LL, respectively) ies, and samples were subjected to Gram-Positive enrichment and general16S rDNA bacterial PCR ampli cation followed by DGGE. We observe different patterns of migration between developmental stages and groups suggesting different bacterial communities. Besides, our DGGE Gram-positive enriched experiments over all samples recovered just three communities in the midgut of wild larvae y samples. To con rm this, we tested a minimum amount of DNA for the absence of actinomycetes in other stages of the y. We could only detect actinomycetes up to a minimum concentration of 0.14 ng of DNA, suggesting that if there are Gram-positive, they are found in undetectable amounts in the samples analyzed by DGGE (with the primers used) as has been reported for taxa of low abundance (González et al. 2011). It has been demonstrated that the detection limit of PCR-DGGE is 104-108 CFU/ml (Ercolini 2004), suggesting that the communities in our samples are below these limits or the primers were ine cient for actinomycetes in this y. Indeed, ampli cation of 16S rDNA for Gram-positive showed smear bands (data not shown) in all y stages, which were subsequently used for cloning.
In general, population index analyses show that adult ies harbor higher diversity than larvae. Speci cally, Shannon index (H') analyses (Table 3) showed that adult wild ies presented the higher bacterial diversity (H'=1.22), as could be expected in a native environment varied diet setting a more diverse group of microorganisms. The wild larvae showed lower diversity (1.07 H') than the adult, as its development is limited within the host fruit. Furthermore, Jaccard index (J) analysis (Table 3) showed that LW and AW share 53% of their bacterial communities, suggesting a bacterial coreconserved community or vertical transmission of bacterial species as observed in C. capitata (Behar et al. 2008).  Regarding mass-rearing ies, larvae and adult samples showed low diversity (H' = 0.94 and 0.95, respectively) than wild ies and shared 63% of their communities. It is worth mentioning that the adult wild ies were collected as adults from mango orchards and not from fruits infested with larvae letting remain in lab conditions until emergence, so the wild gut microbiome was better represented. Comparison of mass-rearing and wild ies showed that larvae samples share 52%, while adult ies share only 36% of their microorganism communities. Since the mass-rearing A. obliqua colony has been maintained for more than 150 generations, this allows us to suggest that it must contain bacterial core microbiota from both natural and mass-rearing environments, which could contribute to the metabolic function of the ies.
Upon bacteria diversity evaluation, we were interested in identifying the species that could be harbored in the y midgut. With this in mind, we cloned, sequenced, and analyzed ribosomal genetic markers from our samples. We obtained 30 bacteria (using general primers) and 23 Gram-positive enriched population sequenced clones; after analysis, we kept 15 bacteria and 3 actinomycetes unique sequences. Thus, the taxonomic classi cation of these sequences was restricted because of the low throughput cloning and sequencing methods.
The phylogenetic analysis of bacteria cloned sequences showed two main groups (Fig. 1)  . Thus, this bacteria species could produce a profound impact on the mass-reared samples with respect to the wild in regards to its tness. Additional to Gram negative bacteria, we identify one Larvae sequence related to the Gram positive Aerococcus viridians and Enterococcus fecalis. All those bacteria species have been differentially described in wild adult's caught, as well as larvae and adult mass-reared ies of Drosophila melanogaster, C. capitata, Bactrocera spp, and A. ludens, respectively ( The mass-rearing adult sequences clustered within metabolically specialized species of bacteria, such as members of the Desulfovibrionaceae family and Lentisphaerae phylum (i.e., Victivallis and Oligosphaera spp). Sulfate-reducing bacteria (Desulfovibrionaceae family) are found in humans (Loubinoux et al. 2002), where they modulate the metabolism of primary fermenter bacteria in the intestine to regulate energy supply. In insects, Desulfovibrionaceae family bacteria are naturally found in the beetle Amblonoxia palpalis (Koneru et al. 2016). Lentisphaerae-isolated bacteria are related to sugar fermentation (Qiu et al. 2013). The presence of most of the adult sequences in this bacteria group suggests that adult laboratory ies develop a specialized microbiota related to the mass-rearing process. In the Gram-positive bacterial group, one mass-rearing adult sequence was related to the Rarobacter species which use torula yeast as a complement to its carbohydrate's metabolism (Shimoi and Tadenuma 1991). Thus, it might play an important metabolic role within the y, since arti cial diets contain different compounds, such as torula yeast (Montoya and Toledo 2010). Altogether, in addition to the irradiation process, the mass-rearing conditions and biological processes within the y could also be responsible for controlling the bacteria load. These results suggest the importance of the microbial species described herein modulating the metabolism of massrearing A. obliqua fruit ies, although their functions during the nutrient assimilation process have yet to be determined. Also, suggest that microbiota and host interactions mediate the proper metabolic conditions to modulate y tness.
Regarding wild samples (Fig. 1, triangles), larvae sequences were related to the Enterobacteriaceae (Escherichia and Kosakonia) family. Kosakonia sacchari is a nitrogen-xing bacterium reported in Saccharum o cinarum L, and uses sugars such as fructose and glucose (Chen et al. 2014). The wild adult sequences were related to Tatumella and Pluralibacter, this last and Enterobacter cloacae were detected in A. ludens (Kuzina et al. 2001).  Table 3). The trend, as in bacterial communities, is that the wild adult exhibits greater diversity than wild larvae and mass-rearing samples, possibly due to a greater variety in their diet. The Jaccard index showed that wild specimens have similar archaea communities (77%), whereas the adult and larvae mass-rearing ies share 83% of their communities. Besides, a comparison between adult and larvae stages also showed that they share more than 70% of their communities. Speci cally, the WA and LA samples share 78%, while WL and LL share 85% of their archaea communities. Archaea are endowed with particular paths to modify metabolic pathways which are related to sugar fermentation (Bräsen et al. 2014) suggesting a role in the host metabolism processing. Overall, we detect the presence of archaea species for the rst time in this Anastrepha species. Our data suggest that archaea may be essential throughout the cycle life of the y and likely could be determined by the mother and diet, as in bacteria.
To gain a better perspective of the archaea species present in the midgut of A. obliqua, we got 29 clean sequences from archaea PCR amplicons. Then, identity was assessed at both archaea and bacteria NCBI databases since primers are ambiguous to bacteria (Coolen et al. 2004). With the gathered information, we construct a phylogenetic tree that discerns archaea and bacteria clades (Fig. 2). Outstandingly, bacterial species detected with archaea-directed primers were different from the species identi ed with bacteria primers (compare Fig. 1 with bacteria clade in 2).
Mass-rearing samples clustered at the archaea clade and one LA sequence clustered at the bacteria clade within the Enterococcus branch. Interestingly, nine samples from mass-rearing larvae were related to Halalkalicoccus species (Halobacteriaceae family), which can grow on fructose and mannose in the laboratory (Xue et al. 2005;Poehlein et al. 2016). Laboratory adult samples were related to strains that use inorganic compounds as an energy source but also organic compounds, such as yeast (i.e., Nitrososphaera spp, and Thermo lum spp) (Tourna et al. 2011;Toshchakov et al. 2015). With basis of the nutritional content of the arti cial diet (including torula yeast) employed for mass-reared ies and the microbial species found, our results suggest that those strains could be gained via the diet, modulating the bio-assimilation process.
In the wild samples, larvae sequences clustered within the Haloterrigena, Natronorubrum, Natrinema, and Haloferax branch (Halobacteriaceae family). These species are related to phosphorus solubilization and can grow in the presence of fructose (Yadav et al. 2015). Interestingly, wild larvae were collected from mangoes, and it is possible that these species could be enriched, as the mango has a high content of fructose and phosphorus. Therefore, these strains could be ingested with the natural diet, or acquired through vertical transmission, contributing to the host's sugar assimilation. Finally, wild adult sequences clustered within the bacteria clade (i.e. Ralstonia, Herbaspirillum, and Ochrobactrum), and no archaea were detected. Isolated Ralstonia and Herbaspirillum strains are associated with copper biosequestration and degradation of chlorophenol, respectively (Im et al. 2004;Yang et al. 2010). In mango crops, copper and chlorophenol are used in distinct formulations to avoiding different diseases caused by fungus and bacteria (Czaplicka 2004;Sarwar 2015). In ies, depending upon the dose, copper compound leads to positive and negative effects.
Positively, copper works by activating superoxide dismutase to regulate oxidative stress, modulating the genotoxic effects of reactive oxygen species and therefore, y survival increases; while copper intoxication leads to the opposite outcome (Matsuo et al. 1997;Arcaya et al. 2013;Southon et al. 2013). Meanwhile, chlorophenol has detrimental outcomes in different organisms (Czaplicka 2004). This suggests that the species harbored in the A. obliqua intestine could modulate y tness by regulating copper and oxidative metabolism.
To the best of our knowledge, this is the rst record of archaea in the digestive tract of A. obliqua. Archaea are important organisms for metabolism in the intestines of insects (Ceja-Navarro et al. 2014). It has also been described that archaea produce metabolites that are used by insects to locate fruits (Piñero et al. 2015). Thus, archaea and the rest of microorganisms could promote food pre-digestion, change the nutritional content, or improve digestion in the y gut, but also suppress the toxic effects of chemical compounds used in crops. Altogether, the results presented here suggest that archaea and bacteria strains, which could be ingested during natural or arti cial feeding, could modulate metabolic pathways in tephritidae through the digestion and assimilation of nutrients. Therefore, the differences between archaea and bacteria specimens detected among our samples can highlight their importance in the midgut.  (Table 1). Despite the efforts we did not obtain clones possible due to the lower concentration of fungi DNA related to it host DNA. Nevertheless, our DGGE results suggest the presence of fungi at A. obliqua at midgut, mainly at wild samples, and that adults have more diversity than larvae.
It is known that the fungal community can play essential roles in insect guts and has been shown to produce toxins that are pathogenic for insects (Kostovcik et al. 2015). As bene cial guests, fungal communities could act as suppliers of organic ingredients, essential vitamins, and enzymes that promote digestion (Vega and Dowd 2005). Fungi could also improve metabolism via sugar fermentation and nitrogen-xing, as well as participate in pheromone production by synthesizing steroids, as has been shown in bark beetles (Klepzig et al. 2009). Some of these functions may occur in the digestive tract of wild larvae and adult ies, which could explain the differences found between wild and laboratory samples.

16S rDNA hypervariable region diversity
Our study aimed to increase our knowledge regarding A. obliqua microbiota. Through this process, we found that some biases must be overcome to determine microbial diversity with the best possible accuracy. These biases start with the selection of DNA extraction methods, primer design, and PCR yield, being determinative to identify the microbiome composition in a sample (Guillén-Navarro et al. 2015; Abellan-Schneyder et al. 2021). We showed that with the use of different primer sets aimed at the same target (diverse hypervariable regions of bacteria and archaea 16S rDNA), it was possible to identify species that have not been previously described in this fruit y. Next-generation sequencing methods using 16S rDNA would allow us to increase the number of species identi ed, but could introduce biases due to different factors, one of which is the use of primers targeted to only one or two of nine 16S rDNA hypervariable regions, which limits species identi cation in complex bacterial populations (Laursen et al. 2017;Bukin et al. 2019). Thus, the phyla Proteobacteria, Actinobacteria, and Deinococcus have been favored as the most abundant in Anastrepha ludens, A. obliqua, A. serpentina, and A. striata through 16S pyrosequencing, with primers targeted to the V3 hypervariable region (Ventura et al. 2018). In this work, we systematically amplify 16S ARN hyper variable regions of V6-V8 to Gram-negative, V1-V8 > V6-V8 (nested) to gram positive (nested), and V1-V6 > V4-V5 (nested) to archaea (Online Resource 1). With this, we observe that V6-V8 hyper variable region captured with primers to gram-positive bacteria, can also identify gram-negative bacteria. Outstandingly, the V4-V5 archaea regions also retrieve blast to specialized bacteria. Despite, other hyper variable regions are used to evaluate microbial diversity (V2-V3), our experimental approach provide important understanding of the microbial composition in this y (Bukin et al. 2019). Our results also broaden our knowledge of the microbiota of A. obliqua with a 28% global abundance of the Archaea kingdom ( Fig. 3 and Online Resource 4, 5).

Conclussion
In this work, we present the inter-kingdom diversity harbored in the midgut of A. obliqua, expanding the intricate microbial crosstalk, which could be related to different biological outcomes (e.g., bio-assimilation) in larvae and adult wild or laboratory ies. It is important to note that the techniques based on the analysis of rRNA genes ampli ed by PCR may not represent a complete and accurate picture of the microbial community. This study is only the beginning of our understanding of the intestinal ecosystem of Anastrepha. We have determined some predominant populations, which could signi cantly modulate the tness and physiology in mass-rearing and wild A. obliqua ies through an inter-kingdom communication of such communities. Archaea and metabolic specialized bacteria were identi ed here for the rst time, suggesting an important role to y tness. Further studies are required to understand how these microorganisms affect metabolic processes, sexual behavior, and other aspects of the y's physiology. This knowledge will eventually improve the mass rearing technology of A. obliqua, and focus on the discovery of microorganisms and enzymes of nutritional interest for fruit ies. Figure 1 Phylogenetic tree of bacteria species identi ed in the intestine of Anastrepha obliqua. Gram-negative and positive bacterial communities are delimited in blue and red, respectively. The 16S ribosomal DNA (rDNA) bacterial clones obtained from wild adult and larvae ies are shown as WL_ (pink triangles) and WA_ (red triangles); the laboratory samples as LL_ (blue light circles) and LA_ (blue circles). The tree (439 positions) was constructed using maximum composite likelihood (MCL) with a Kimura 2-parameter model. The percentage higher than 70% of 1000 bootstrap resampling is shown next to the branches. Evolutionary analyses were conducted using MEGA6 Figure 2 Phylogenetic tree of archaea communities identi ed in the intestine of A. obliqua. Two main clades can be seen, archaea (green) and bacterial (blue) communities. The 16S rDNA bacterial clones obtained from wild adult and larvae ies are shown as WL_ (pink triangles) and WA_ (red triangles); the laboratory samples as LL_ (blue light circles) and LA_ (blue circles). DAMBE software was employed to merge equal sequence clones. The neighborjoining method to a matrix of pairwise distances estimated using the MCL approach with a Kimura 2-parameter model was used to construct the tree (406 positions). The percentage higher than 70% of 1000 bootstrap resampling of trees in which the associated taxa clustered together is shown next to the branches. Evolutionary analyses were conducted using MEGA6.

Figure 3
Microbial community pro le identi ed in the intestine of A. obliqua. The 16S rDNA bacterial, actinomycete, and archaea clones obtained from wild and laboratory adult and larvae ies were curated, merged, and analyzed in Geneious software to determine 16S biodiversity. Most of the recovered sequences (~85%) were relative to bacteria showing three main clades (proteobacteria, rmicutes, and actinomycetes) ~25% were relative to Archaea