Yersinia massiliensis ( Enterobacteriales : Enterobacteriaceae ) in the host Anaphes nitens ( Hymenoptera : Mymaridae ) : first report of association with insects Yersinia massiliensis ( Enterobacteriales : Enterobacteriaceae ) no hospedeiro Anaphes nitens ( Hymenoptera : Mymaridae ) : primeiro relato de associação com insetos

The use of the egg parasitoid Anaphes nitens (Girault, 1928) (Hymenoptera: Mymaridae) is the main management strategy for the Eucalyptus spp. defoliator beetle, Gonipterus platensis (Marelli, 1926) (Coleoptera: Curculionidae) (Souza et al., 2016). This parasitoid is originally from Australia (Mapondera et al., 2012), and it has been successfully introduced in South Africa, Spain and California (United States of America) (Cordero-Rivera et al., 1999; Hanks et al., 2000) but with low efficiency in some regions of Portugal, Chile and Brazil (Reis et al., 2012; Gumovsky et al., 2015; Souza et al., 2016). The reasons for the low parasitism rates in these countries are not fully understood and some authors attribute it to the seasons, altitude or host-parasitoid incompatibility (Reis et al., 2012; Mapondera et al., 2012). Secondary endosymbiont bacteria can reduce fitness (Zug and Hammerstein, 2018) and parasitism rates (Furihata et al., 2015), increase susceptibility to Abstract Endosymbiont bacteria can affect biological parameters and reduce the effectiveness of natural enemies in controlling the target insect. The objective of this work was to identify endosymbiont bacteria in Anaphes nitens (Girault, 1928) (Hymenoptera: Mymaridae), the main natural enemy used to manage Gonipterus platensis (Marelli, 1926) (Coleoptera: Curculionidae). Genomic DNA from six A. nitens populations was extracted and polymerase chain reactions (PCR) were performed with the primers to detect endosymbiont bacteria in this insect. The PCR products were amplified, sequenced, and compared with sequences deposited in the GenBank for the bacteria identification. All A. nitens populations had the bacterium Yersinia massiliensis (Enterobacteriales: Enterobacteriaceae). This bacterium was originally described as free-living, and it is associated with and composes part of the A. nitens microbiota. This is the first report of Y. massiliensis in an insect host.


Introduction
The use of the egg parasitoid Anaphes nitens (Girault, 1928) (Hymenoptera: Mymaridae) is the main management strategy for the Eucalyptus spp. defoliator beetle, Gonipterus platensis (Marelli, 1926) (Coleoptera: Curculionidae) (Souza et al., 2016). This parasitoid is originally from Australia (Mapondera et al., 2012), and it has been successfully introduced in South Africa, Spain and California (United States of America) (Cordero-Rivera et al., 1999;Hanks et al., 2000) but with low efficiency in some
Palavras-chave: controle biológico, parasitoide de ovo, microbiota, entomologia molecular.  (Van Nouhuys et al., 2016) and alter the structure and composition of parasitoid communities (Rothacher et al., 2016;Monticelli et al., 2019). It is important to know the host microbiota to evaluate its interference in parasitoid efficiency in biological control programs (Almeida et al., 2010). However, there are no reports of endosymbiont bacteria in A. nitens. The objective of this work was to identify the presence of endosymbiont bacteria in the A. nitens host populations in Brazil.

Materials and Methods
Egg masses of G. platensis were collected in commercial Eucalyptus spp. plantations in the municipalities of Botucatu, Itararé, Itatinga, Lençóis Paulista and Pratânia in the state of São Paulo and Aracruz in the state of Espírito Santo, Brazil. They were kept in acrylic plates (5 cm diameter and 1 cm high) in biochemical oxygen demand (BOD) incubator chamber at 25 °C and a photoperiod of 12 hours until the emergence of A. nitens adults. The emerged insects were collected and preserved at 4 °C.
Genomic DNA from 50 adult insects per A. nitens population was extracted following the Chelex 100 protocol (Walsh et al., 1991). These insects were washed in 70% alcohol, macerated and homogenized in a solution of 80 μL of Chelex 100 resin (Bio-Rad Laboratories, USA) 10% and 8 μL of proteinase K (20 μg/mL) (Bioline, USA). Samples containing the DNA of the A. nitens populations were transferred to an Infinigen Thermal Cycler (model TC-96CG) and incubated at 95 °C for 20 minutes.
Egg capsules of G. platensis from laboratory rearing, up to 24 hours old and free of parasitism, were analyzed as the control. These capsules were immersed in distilled water for 1 hour, with a subsequent sampling of 35 eggs and the excrement layer of five egg capsules. The genomic DNA extraction procedure was the same as for A. nitens samples.
The 23S region was amplified with the primers of the secondary endosymbiont Arsenophonus, w i t h p o l y m e r a s e c h a i n r e a c t i o n ( P C R ) 5'-CGTTTGATGAATTCATAGTCAAA-3 '(Forward) and 5' GGTCCTCCAGTTAGTGTTACCCAAC-3 '(Reverse) (Thao and Baumann, 2004). A PCR mix containing 12.5 μL of Taq DNA Polymerase (NeoBio), 7.5 μL water milliQ, 1.0 μL of each primer and 3.0 μL of DNA, totaling a volume of 25 μL, was used in each of the population samples. PCR reactions were performed under the following conditions: initial denaturation temperature at 95 °C for 2 min, followed by 30 cycles each of 95 °C for 30 sec, 58 °C for 30 sec, 72 °C for 1 min. and a final extension of 72 °C for 5 min. A molecular marker 100pb (Norgen) and 1% agarose gel, visualized in a UV Light Transilluminator (Major Science), were used. PCR products were purified using a "PCR Purification Kit" (Cellco Biotec, Brazil). The amplified DNA fragments of the bacteria were sequenced by the Sanger DNA Automatic Sequencer (Model: ABI 3500-Applied Biosystems), at the Institute of Biotechnology (IBTEC/UNESP) in Botucatu and compared with the sequences deposited in GenBank, using the BLASTn program (Blast, 2020).
The nucleotide sequences obtained were analyzed using the software Geneious v11.1.5 and compared to a dataset composed of other accessions retrieved from the GenBank. The sequences were aligned using MAFFT v7.222 within the Geneious v.11.1.5 software, and phylogenetic analysis was performed using MrBayes 3.2.2 (Ronquist and Huelsenbeck, 2003). Two independent runs were conducted simultaneously using 10 million generations and excluding 25% from the resulting tree as burning. Phylogenetic tree was visualized, edited and rooted using FigTree v1.4.4 (FigTree, 2020).

Results
BLASTN analysis of the 600 bp amplicon resulted in high identity (98% identity with the accession number CP028487-1) with the bacterium Yersinia massiliensis (Enterobacteriales: Enterobacteriaceae).
This bacterium was found in all A. nitens populations and it was absent in G. platensis eggs and excrement layer of the egg capsules ( Figure 1). Phylogenetic analysis grouped the sequences of this bacterium in a single sub-clade including samples from all localities (Figure 2).

Discussion
The presence of the bacterium Y. massiliensis in all populations of A. nitens includes it in the composition of the microbiota of this insect host. The Y. massiliensis DNA sequencing from the primer developed for the endosymbiont Arsenophonus may be due to the amplification of the 23S gene region in a non-specific way (Rekha et al., 2006). Yersinia massiliensis was originally described as a freshwater free-living gramnegative coccobacillus (Merhej et al., 2008) and has been found in aquatic and food environments in Brazil and Argentina (Souza et al., 2011) as non-pathogenic (Thomas et al., 2018).
Some bacteria in the Enterobacteriaceae family may be free-living bacteria or secondary endosymbionts (Renoz et al., 2019), as the genus Hamiltonella, Regiella, Serratia and Yersinia (Kikuchi, 2009). The composition of the insect microbiota varies with the host, from a few dozens in Lepidoptera (Paniagua Voirol et al., 2018) to hundreds of species in termites (Brune and Dietrich, 2015). Insects can acquire free-living bacteria and may establish mutual relations with them (Sudakaran et al., 2017). The host diet is the main impact factor in the available resources for the microbiota and, consequently, defines the microorganism species surviving in its interior (Foster et al., 2017).
The genus Yersinia currently contains 15 species, Y. enterocolica, Y. pseudotuberculosis and Y. pestis are pathogenic to humans and some vertebrates, Y. entomophaga is pathogenic to insects, and the others are considered non-pathogenic opportunistic bacteria (Merhej et al., 2008;Hurst et al., 2011;Souza et al., 2011, Reuter et al., 2014. Yersinia massiliensis was found in A. nitens collected in areas with a high parasitism rate (State of Espirito Santo) or low parasitism rate (State of São Paulo) (Souza et al., 2016) by this natural enemy on G. platensis. Therefore, it is unlikely to be the cause of parasitism differences in Brazil.
The association of Y. massiliensis with the parasitoid A. nitens represents the first report of this bacterium in an insect host.

Acknowledgements
To the Brazilian institutions "Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)", "Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Finance Code 001), "Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)" and "Programa Cooperativo sobre Proteção Florestal/PROTEF do Instituto de Pesquisas e Estudos Florestais/IPEF" for financial support. David Michael Miller, a professional editor and proofreader and a native English speaker has reviewed and edited this article for grammar, punctuation, spelling, structure, word choice, and readability.