Bacteria in digestive tract of Simuliidae (Insecta: Diptera) larvae from the Brazilian Amazon

Simuliidae are known to be vectors of tropical diseases, as well as the transmission of viruses, bacteria and protozoa. The objective of the study was to isolate and identify bacteria from the digestive tract of larvae from breeding sites in several municipalities in the Amazon, especially in the States of Amazonas and Rondônia. Larvae were collected using tweezers and stored in sterile tubes under refrigeration. In the laboratory, a total of 210 larvae were dissected under aseptic conditions and then bacterial isolation, multiplication and characterization were carried out. Specific identification was based on the 16S ribosomal DNA method, complemented with biochemical tests (BBL Crystal, BBL and Bactray System, Laborclin). A total of 58 bacterial isolates were obtained and the representative genera in the larvae of 7 Simuliidae species analyzed were Bacillus, Staphylococcus, Haphinia , Chromobacterium and Escherichia. Knowledge about the bacterial diversity of insects can contribute both to a better understanding of host-microorganism relationships and to the search for bacterial species for use in biological control programs for insect vectors of diseases or agricultural pests.


INTRODUCTION
Carl Woese (Woese, et al. 2000) was the first to intensively apply molecular methodology to the phylogeny of microorganisms.With this, he revolutionized the science of microbiology, discovering the existence of 3 domains at the origin of life (Bacteria, Eukaria and Archaea).
Subsequent studies confirmed the suspicion that only about 5% or less of the microorganisms existing in the biosphere are amenable to cultivation in the laboratory, but can be identified by molecular methods (Ward et al. 1990, Pace, 1997and Lee et al. 2021).
Prokaryotes have highly conserved regions and the analysis of this material is quite efficient when using specific regions for each bacterial taxon.The use of primers complementary to the regions amplifies the gene region, generating a nucleotide sequence that can be compared to Genbank.(NCBI).This method opened up enormous perspectives for the analysis of species evolution, thus allowing phylogenetic and taxonomic analyses.The 16S rDNA gene was the target of the study.The amplification product was cloned and sequenced according to the technology established at the Laboratory of Molecular Genetics and Genomes at the University of Vale do Parnaíba (Univap).
Insects are the most abundant animals on Earth, and the microbiota within their guts play important roles by engaging in beneficial and pathological interactions with these hosts (Yun et al. 2014).
Bacteria can be transmitted horizontally from the environment to host insects along with food flow, and the digestive tract of insects provides ideal habitats and abundant nutrients for bacterial colonization (Graf, 2016).
Brazilian Journal of Animal and Environmental Research, Curitiba, v.6, n.4, p. 3328-3338, out./dez., 2023 Several studies have investigated the microbiota of Simuliidae and Culicidae larvae in the search for new strains of insecticidal activity and for biological control of insect pests or even for purposes in the pharmaceutical industry (Ríos-Velásquez & Hamada 2002, Alencar et al. 2003, Pereira et al. 2005, Lobo et al. 2008, Alencar et al. 2017, Lee et al. 2021).Cheethan (1985) speculates that less than 1% of all microorganisms have been identified and characterized.However, recent studies have already shown the applicability of substances produced by microorganisms, such as some toxins produced by various species of bacteria that have high insecticidal, acaricidal and nematicide action.Bacteria Bacillus sphaericus is notably known for its use in mosquito control, as well as B. Turingiensis var.Israelensis has been researched and used in mosquito control (ALVES, 1998).Thus, the possibility of discovering new substances in microorganisms host associations that can contribute to biological control studies of tropical endemic vector insects.
The objective of the study was to isolate and identify bacteria from the digestive tract of larvae from breeding sites in several municipalities in the Amazon, especially in the States of Amazonas and Rondônia.

MATERIAL AND METHODS
This study was conducted in September to October 2003 and May to Juny 2004 in different localities within the municipalities of Manaus, Presidente Figueiredo, Amazonas and Rondônia (Table I).These streams have many characteristics in common: all flow through yellow latosol soil and are located in areas that have been disturbed due to the opening of highways.
Collection of Simuliids-Approximately 30 black fly larvae were collected using forceps, placed in containers with sterile distilled water, and stored in under refrigeration before being dissected in the laboratory.Morfological characters used for species characterization were the same as those used in conventional studies of Simuliidae systematics (e.g.Crosskey, 1990, Hamada, 1998, Hamada & Adler, 2001) Isolation of bacteria-These insects were sterilized following the methodology described by Alves (1998) by consecutives washings in distilled water, and passing the larvae in 70% alcohol for II vertical laminar flow aseptic chamber.For each Simuliidae species pools gut samp les from 30 insects were macerated in 500 µl of sterile culture media Luria Bertani (LB).An aliquot of 100 μl was inoculated on Petri dishes in triplicate containing the following culture media: LB (Luria Bertani agar) and MC (MacConkey agar).The plates were incubated at 37º C and examined for 18 a 24h.Bacterial load was quantified by determine the colony forming units (CFU).
Sample identification -Bacteria were scraped off from the agar surface and stained with Gram´s reagent.Samples were also run through the biochemical kits Bactray I, II, III (Laborclin) and BBL Crystal.Test results were analysed using Bactray software version 2.1 and BBL Crystal for 2 minutes, followed by 30 cycles of 95ºC for 1 minute, 61ºC for 40 seconds and 72ºC for 2 minutes.At the end of the last cycle, final elongation was performed at 72 ºC for 5 minutes.The result was analyzed in an electrophoresis tank with 0.8% agarose gel impregnated with ethidium bromide.
For sequencing, the PCR amplification product was purified using the Concert Rapid PCR Purification System from Gbco BRL (Life Technologies).
Brazilian Journal of Animal and Environmental Research, Curitiba, v.6, n.4, p. 3328-3338, out./dez., 2023 The sequences of the rDNA gene were determined by the enzymatic method of terminating and elongating the triphosphate dideoxyribonucleoside chain using an automatic sequencer.

RESULTS AND DISCUSSION
A total of 58 bacterial isolates were obtained from the intestinal macerate of seven species  were also identified (Oliveira et al., 2000).from the North region.They found that the predominant species in the samples were Bacillus sphaericus and Bacillus thuringiensis and a great ecological diversity among the Brazilian regions analyzed.This also mentions that although hundreds of species of bacteria associated with insects are known, few are those that can be used for biological control of agricultural pests and vectors of tropical diseases, highlighting the importance of searching for new strains or genes with characteristics of pathogenicity.
The results obtained in the present research allowed the establishment of a bank of strains to be used for experimental tests both for the biological control of insect vectors of tropical diseases and for bioprospecting studies, with a view to identifying bacteria of biotechnological importance and microbial control.As an example, the bacterium Chromobacterium violaceum is classified as beta proteobacterium, gram-negative, facultative anaerobe, found in the soil and waters of tropical and subtropical ecosystems around the world.The species produces, through secondary metabolism, several indole pigments derived from tryptophan.Violacein and deoxyviolacein are the most abundant pigments produced by C. violaceum (Pitlovanciv et al. 2006).
Bacillus thuringiensis (Bt) is a microbial control agent, whose main characteristic is the production of insecticidal crystals during its sporulation.The entomopathogenic properties of this bacterium have driven research to select Bt isolates with toxic activity for different insect species (Schnepf et al. 1998).In the present study, this species was identified in the digestive tract of Simuliidae larvae.
In addition to the results demonstrating the presence of species of bacteria that have potential for use in biological control, such as bacilli, other species considered pathogenic to humans were also identified in the samples, such as Eschericia coli, Streptococcus spp.and Staphylococcus spp.
Bacillus cereus itself can also cause food poisoning by causing vomiting and/or diarrhea.Bacillus cereus can cause two distinct forms of gastroenteritis.Most of its strains are capable of producing metabolites which are related to their virulence mechanism (Granum et al. 1997).Staphylococcus species, which are cocci grouped in the form of clusters, may be related to intestinal and pyogenic infections (Becker et al. 2014).
In Brazil, research on the microbiota of insects is still scarce, with the majority covering studies of a purely taxonomic nature.Our study demonstrated that several species of microorganisms can live in associations within the intestine of Simuliidae larvae, including some with pathogenic potential, confirming the importance of identifying and reporting the presence of these microorganisms in all possible types of habitat and justifying differentiated studies in entomology.
Based on the results of the identification of bacteria associated with Simuliidae larvae, it was possible to increase the collection of cultures of microorganisms associated with insects currently stored at the National Institute for Amazonian Research/INPA, providing an increase in the number of strains identified and which are being studied for application in industry or in control programs for insect vectors of diseases or pests in agriculture.
Considering arthropods, insects are the most abundant and most diverse group.This group has reached a high level of specialty to conquer various niches given the enormous availability of food, ideal conditions for reproduction and development such as high temperatures, high humidity, abundant water collections, in addition to low intra-specific competition.Especially the insect vectors that associated and adapted to human conditions found in this association excellent conditions that favored the success of their species.In this situation, some parasites (viruses, protozoa and filaria) also took advantage of their spread, which has caused huge epidemics.In view of this, studies on the diversity of bacteria associated with immature stages of Insecta are important as they can provide information on microorganism-host relationships and thus provide specific action for the biological control of target species and also obtain new drugs contributing to human and animal health. .
Despite the difficulties faced with the lack of resources for research and the high value of materials for analysis, partnerships are important in overcoming limitations to achieve the proposed objectives.

CONCLUSIONS
The research contributed for knowledge of the microbial diversity of various species of Simuliidae of the Amazon with species of interest to biocontrol and screening of substances.
Bacteria strains were identified by molecular analysis which contributes to the increase of the INPA microorganism collection.
Mind.Wirtz staining was used for the identification of spores.Biochemical, cytological and physiological characteristics according to Bergey´s Manual of Determinative Bacteriology were used in the classifications(David et al., 2001).Representative cultures were preserved in LB soft agar at 4ºC.Long term storage was done in LB broth containing 15% glycerol at -80ºC as part of the culture collection of Instituto Nacional de Pesquisas da Amazônia (INPA).Molecular analyses-representative clones of bacteria obtained after cultivation were used for the DNA extraction steps.For this, 5 representative bacterial strains were chosen and added to 1mM EDTA solution.DNA extraction was then performed according toOgram et al. (1987), DNA purification was subsequently required using a solution of CTAB and Chloroform and isoamyl alcohol (24:1) followed by precipitation with a solution of 13% polyethylene glycol in 1.6 M NaCl, 4 M ammonium acetate and 100% ethanol.The DNA was washed with 70% ethanol.The precipitate was dissolved in 200µL of TE and the DNA was used for polymerase chain reaction PCR.Using the universal primers D88: 5' GAG AGT TTG ATY MTG GCT CGA 3' and E94: 5' GAA GGA GGT GWT CCA RCC GCA 3'(Pasteur et al., 2001)  we amplifield the gene that codes for the ribosomal 16S bacterial RNA generating a fragment of about 1500bp of genomic DNA.For archaea, the following generic primers were used, 519F 5' CAG CMG CCG CGG TAA TWC 3' 18 mer (W= A/T; M=A/C) and 1406R 5'ACG GGC GGT GTG TRC 3' 15 mer (R =A/G)(Cowen et   al., 2003)  with the expected size of the obtained fragment being 870bp, which amplify specific sequences from certain bacterial groups.For PCR, an initial denaturation was carried out at 95ºC

(
2001) isolated, as the first record for Brazil, entomopathogenic bacteria from Simulium larvae and adults in the states of São Paulo and Rio de Janeiro.These identified 18 strains of Bacillus thuringiensis and 1 of Bacillus sphaericus, and these isolates were toxic to larvae of Aedes aegypti and Culex quinquefasciatus.Snooddy & Chipley (1971) observed the presence of 5 bacterial isolates in the digestive tract of Simulium underhilli in the United States, including: Micrococcus sp., Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa and Aeromonas hydrophila.The associations between Simulid larvae and bacteria are mainly related to the larvae's characteristics of filtering various particulate materials suspended in water.Fermenting and nonfermenting bacteria appear to be the most abundant in the microbiota of the larvae analyzed in the research.The genus Bacillus presented greater frequency in the digestive content of Simuliidae larvae in the breeding sites studied, forming part of the larvae's diet.In a study on the identification of bacteria from the digestive tract of female Lutzomyia longipalpis, non-fermenting gram negative species were predominant, being represented by Acinetobacter lwoffii, Stenotrophomonas maltophilia, Pseudomonas putida and Flavimonas orizihabitans.Gram-negative fermenting bacteria were also found: Enterobacter cloacae and Klebsiella ozaenae.Gram-negative bacteria such as Bacillus thuringiensis and Staphylococcus spp.