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Applied and Environmental Microbiology, January 2009, p. 547-550, Vol. 75, No. 2
0099-2240/09/$08.00+0     doi:10.1128/AEM.01665-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Detection of Wolbachia Bacteria in Multiple Organs and Feces of the Triatomine Insect Rhodnius pallescens (Hemiptera, Reduviidae){triangledown}

C. I. Espino,1 T. Gómez,1 G. González,1,2 M. F. Brazil do Santos,2 J. Solano,2 O. Sousa,1 N. Moreno,1 D. Windsor,3 A. Ying,1 S. Vilchez,2 and A. Osuna2*

Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad de Panamá, República de Panamá,1 Grupo de Bioquímica y Parasitología Molecular, Instituto de Biotecnología, Universidad de Granada, Campus Universitario de Fuentenueva, 18071 Granada, Spain,2 Smithsonian Tropical Research Institute, Apdo. 0843-03092, Panamá, República de Panamá3

Received 18 July 2008/ Accepted 12 November 2008


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ABSTRACT
 
At least two types of Wolbachia bacteria were detected in wild and insectarium-raised Rhodnius pallescens, a natural vector of Trypanosoma cruzi and Trypanosoma rangeli. Wolbachia was detected in all the organs and tissues studied and in the feces, and this provided a methodological advantage for determining the presence of this endosymbiont in this host, obviating the need to kill the specimens. The occurrence of trypanosomatids in wild individuals was also studied.


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INTRODUCTION
 
Wolbachia is an obligate intracellular bacterium (18) that is present in 20 to 75% of insect species (3, 14, 33, 38, 41, 42). This bacterium was first described in 1924 in mosquitoes (Culex pipiens) (12, 13) and was initially classified as Rickettsia sp. (9, 33). Wolbachia displays a tropism for the reproductive tissue of its hosts and is transmitted vertically from insect to insect through the ovules, while interspecific transmission appears to occur horizontally with the possible help of parasitoids (1, 3, 6, 17, 19, 33, 43). Despite the fact that infected insects show no pathological signs, the presence of Wolbachia can result in diverse reproductive alterations in its hosts, including parthenogenesis, feminization, male killing, and unidirectional or bidirectional cytoplasmic incompatibility (33, 35, 38, 44).

The relationship between Wolbachia and its arthropod hosts ranges from mutualistic to parasitic, which makes it all the more interesting and necessary to ascertain the exact nature of the interaction between particular symbionts and their hosts (40). Wolbachia isolates have been found in numerous disease-carrying insects, such as Culex (12, 13), Aedes (27), Glossina (2, 26, 39), Phlebotominae (16), Cimex (28, 29), Ctenocephalides felis (11), and Tunga penetrans (10). It also occurs in parasitic nematodes, such as Onchocerca volvulus, is responsible for the inflammatory reaction that induces blindness (22, 24), and has been detected in Brugia malayi (34, 36). Wolbachia has recently been found in Angiostrongylus cantonensis, a nematode that is not related to filarias (37).

The sylvatic triatomine Rhodnius pallescens is considered the most important vector of the trypanosomatids Trypanosoma cruzi and Trypanosoma rangeli in the neotropics. Its capacity to invade houses located near its natural habitat, the royal palm tree (Attalea butyracea), and to transmit T. cruzi and Chagas' disease to humans has been well documented in Panama (4, 23, 32). Wolbachia was previously found in only one individual of R. pallescens that was described in a list of Panamanian species (41). However, information on the occurrence of this bacterium and its distribution in the organs of its hosts is not available. In this work we examined whether there is any correlation between the presence of this endosymbiont and the presence of the parasites T. cruzi and T. rangeli.

In this study we examined a total of 72 individuals of the triatomine insect R. pallescens; 27 of these individuals were collected from their natural habitat, and 45 were obtained from an insectarium. Wild specimens were collected in regions of the Republic of Panama where Chagas' disease is endemic (Table 1). Insectarium specimens were obtained from the Instituto Conmemorativo Gorgas de Estudios de la Salud and from the Centro de Investigaciones Parasitarias de la Universidad de Panamá.


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  		TABLE 1. Occurrence of trypanosomes and Wolbachia in R. pallescens individuals collected from several Panamanian districts

Each specimen was dissected, and gonads, salivary glands, and intestines were extracted under sterile conditions. The posterior intestine, rectal ampolla, and salivary glands from wild triatomines were homogenized, and any trypanosomes present were observed with a microscope or cultured in Grace medium to facilitate detection of T. cruzi and T. rangeli (20, 21). T. cruzi and T. rangeli were also detected by PCR, as previously described (7, 45, 46).

The presence of Wolbachia was detected in each organ by PCR using specific primers for 16S rRNA and wsp genes, as previously reported (42, 44). Standard reaction mixtures (final volume, 10 µl) contained 0.5 µl of the template DNA (extracted with a Qiagen DNeasy tissue kit) plus 0.08 µl of deoxynucleoside triphosphates (25 mM), 0.5 µl of the forward and reverse primers (10 µM), 0.1 µl of Taq polymerase (5 U/µl), 0.5 µl of MgCl2 (50 mM), and 0.4 µl of dimethyl sulfoxide (5%).

The integrity of the total DNA extracted was verified by amplification of the 28S rRNA gene as previously described (5), and DNAs from Nasonia that was positive and negative for Wolbachia (kindly provided by J. Werren) were used as controls.

The results of the screening analysis of the 27 wild triatomines are shown in Table 1. This analysis revealed that 56% of the triatomines were infected with T. cruzi and 25% of the triatomines were infected with T. rangeli. Simultaneous infection with T. cruzi and T. rangeli was also detected in 12% of the specimens. The presence of T. cruzi in the wild insects indicates that the risk of Chagas' disease in humans was elevated in the areas where insects were captured.

PCR analysis with probes for the wsp gene detected the presence of Wolbachia in the gonads and salivary glands of 100% of the insects, while PCR analysis with the primers specific for 16S rRNA detected Wolbachia in 95.9% of the cases. As recommended by Duron and Gavotte (8), the two pairs of primers were used to rule out the possibility of false negatives.

The analysis of specimens from the insectarium produced very different results. Only 51.0% of the insects were positive for Wolbachia with both probes (Table 2). Of the positive insects, 51.0% were positive when the gonads were tested, 44.4% were positive when the salivary glands were tested, and 94.0% were positive when the intestine was tested.


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  		TABLE 2. Occurrence of Wolbachia in insectarium specimens of R. pallescens, as determined by PCR

Given Wolbachia's presence in all of the wild triatomines collected, it seems that the presence of the endosymbiont does not influence the susceptibility of the insects to infection by the parasitic protozoan T. cruzi, the etiological agent of Chagas' disease, and T. rangeli, for which these insects are natural hosts.

Wolbachia is vertically transmitted by oocyst infection and in this way maintains a high incidence in arthropods (33). Its presence has been reported in other tissues, including nerve tissue or hemocytes (25). In order to determine the degree of Wolbachia infection in organs of R. pallescens insects other than the organs used in the screening analysis, an insectarium specimen of R. pallescens was dissected to extract the hemolymph, the musculature, the Malpighian tubules, and the intestine. Each organ was tested for the presence of Wolbachia with specific primers for 16S rRNA and wsp genes. All PCRs were positive, indicating that the bacterium was distributed throughout the tissues of the insect and was not restricted to the digestive tract and gonads.

The presence of Wolbachia both in the salivary glands and in the intestine might be explained by the coprophagous and cannibalistic habits of the insects in the early phases of their development, when they acquire the symbionts essential for their development (30, 31). This could also be the mechanism that transmits and spreads the endosymbiont among triatomines. To verify that Wolbachia is present in the digestive products of the triatomines, feces were collected after an insectarium specimen was fed, and the feces were probed with the 16S rRNA and wsp gene primers. Both amplification reactions were positive. The fact that Wolbachia can be detected in the feces of triatomines makes it unnecessary to neutralize the insects in order to determine the presence of this endosymbiont, which is an important methodological advantage.

In order to characterize the Wolbachia strain present in R. pallescens, several PCR products were sequenced. The sequence of the 16S rRNA gene obtained from feces of an insectarium specimen was 99 to 100% identical to the sequences of a Wolbachia endosymbiont of Pseudolynchia canariensis (accession no. DQ115537) and other unculturable bacteria obtained from insects, such as the cat flea C. felis (accession no. EF121347), the ant lion Myrmeleon mobilis (accession no. EF121347, DQ068883, and DQ068882), and the fruit fly Drosophila melanogaster (accession no. DQ981371, DQ981358, and DQ981347). The same 16S rRNA sequence was found in the feces of a wild Rhodnius specimen collected in Chuzo (Table 1). The wsp gene sequence from this wild individual was compared with the sequences in the database constructed and maintained by K. A. Jolley and L. Baldo (Wolbachia MLST Databases [http://pubmlst.org/wolbachia/]). The results of this comparison showed that the level of similarity with allele 92 in the database was 96.10%. Given that Jolley and Baldo consider a single difference in the nucleotide sequence an indication of different alleles, it appears that the strain of Wolbachia present in the triatomines is a novel strain and is not included in this database.

The fbpA gene was also amplified (15) from gonads of nine wild specimens positive for Wolbachia and sequenced (Table 1). Direct observation of the DNA chromatograms revealed superimposed peaks at 16 different positions. These results were interpreted as showing that R. pallescens was infected by at least two Wolbachia strains. Surprisingly, an analysis of the sequence of the fbpA gene from three insectarium specimens (Table 2) revealed only four superimposed peaks. Although insectarium and wild triatomines were collected from different areas of Panama, superimposed peaks were found at the same positions for both groups. A possible explanation for this observation is that both insect groups were infected by the same Wolbahcia strains and at least one of the strains was cured when triatomines were raised in laboratory colonies. This result strongly indicates that the vertical transmission of Wolbachia could be affected in insects raised under laboratory conditions. Some factors that alter the presence and spread of Wolbachia in insects have been described previously (33). In our case the exposure to high temperatures, the immune response of the vertebrate used for laboratory feeding, or genetic factors of the host could be relevant factors. It would be interesting to study the effect of removing triatomines from their natural habitat and raising them in laboratories on the development or spread of this endosymbiont.

Study of the presence of Wolbachia in triatomines opens up a new area of research and the possibility of using this endosymbiont to manipulate the reproduction of these insects that are responsible for the vectorial transmission of Chagas' disease.


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ACKNOWLEDGMENTS
 
We thank J. Werren of the University of Rochester for his advice and Instituto Conmemorativo Gorgas de Estudios de la Salud and Centro de Investigaciones Parasitarias de la Universidad de Panamá for providing the insectarium triatomines. We also thank J. Trout for revising the manuscript.

Susana Vilchez received a grant from the Programa Ramón y Cajal (MEC, Spain, and EDRF, European Union). We thank the Spanish Agency for International Co-operation for covering the travel expenses of the research team (grants A/5115/06 and A/8187/07).


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Biotechnology, University of Granada, Campus Universitario Fuentenueva, E-18071 Granada, Spain. Phone: 34-958244163. Fax: 34-958243174. E-mail: aosuna{at}ugr.es Back

{triangledown} Published ahead of print on 21 November 2008. Back


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Applied and Environmental Microbiology, January 2009, p. 547-550, Vol. 75, No. 2
0099-2240/09/$08.00+0     doi:10.1128/AEM.01665-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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