1 Multi-organ and fecal detection of Wolbachia in triatomine Rhodnius pallescens 2 (Hemiptera, Reduviidae C

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AEM Accepts, published online ahead of print on 21 November 2008 Appl. Environ. Microbiol. doi:10.1128/AEM.01665-08

1 Multi-organ and fecal detection of Wolbachia in triatomine Rhodnius pallescens 2 (Hemiptera, Reduviidae)

3

11 19 9 9 1

4 C. I. Espino, T. Gomez, G. Gonzalez, ' M. F. Brazil do Santos, J. Solano, O. Sousa, 5 N. Moreno,1 D. Windsor,3 A. Ying,1 S. Vilchez,2 and A. Osuna.

6

7 * Departamento de Microbiologia y Parasitologia, Facultad de Medicina, Universidad i 8 Panama, Republica de Panama.

9 2 Grupo de Bioquimica y Parasitologia Molecular, Instituto de Biotecnologia, 10 Universidad de Granada, Campus Universitario de Fuentenueva, 18071 Granada, Spain.

11 3 Smithsonian Tropical Research Institute, Apdo. 0843-03092, Panama, Republica de 12 Panama.

13 Running title: Wolbachia in triatomines.

15 Journal Section: Invertebrate Microbiology 16

17

18 * Corresponding author, present address: Professor Antonio Osuna, Institute of 19 Biotechnology. University of Granada. Campus Universitario Fuentenueva. E-18071 20 Granada. Spain. Telephone: 34-958244163. Fax: 34- 958243174. E-mail: [email protected] 21

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1 ABSTRACT

2 The presence of at least two types of Wolbachia, were detected in wild and insectarium 3 Rhodnius pallescens, natural vector of Trypanosoma cruzi and T. rangeli. Wolbachia was 4 detected in all the organs and tissues studied and in the feces, which represents jfr 5 methodological advantage when determining the presence of the endosymbiont in 6 host, obviating the need to neutralize the specimen. The occurrence of trypanosomati 7 wild individuals was also studied

8

9 Key words: wild triatomines; insectarium triatomines; Wolbachia wsp; Chagas' disease;

10 Rhodnius pallescens; Trypanosoma cruzi; Trypanosoma rangeli 11

12 Wolbachia is an obligate intracellular bacterium (18) present in some 20 to 75% of insect 13 species (14, 3, 33, 38, 42, 41). This bacterium was first described in 1924 in mosquitoes 14 (Culex pipiens) (12, 13) and initially classified as Rickettsia sp. (9, 33). Wolbachia 15 displays a tropism for the reproductive tissue of its hosts, transmitting itself vertically 16 from insect to insect through the ovules, while interspecific transmission appears to occur 17 horizontally with the possible help of parasitoids (3, 33, 1, 17, 19, 6, 43). Despite the fact 18 that infected insects show no pathological signs, the presence of Wolbachia can provoke 19 diverse reproductive alterations in the host, including parthenogenesis, feminization, male 20 killing and unidirectional or bi-directional cytoplasmic incompatibility (33, 35, 38, 44).

21

22 The relationship between Wolbachia and its arthropod hosts ranges from mutualistic to 23 parasitic, which makes it all the more interesting and necessary to ascertain the exact

1^

ilar bacterium (18}

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1 nature of the interaction between particular symbionts and their hosts (40). Wolbachia 2 have been found in numerous disease-carrying insects, such as Culex (12, 13), Aedes 3 (27), Glossina (39, 2, 26), Phlebotominae (16), Cimex (28, 29), Ctenocephalides felis (11) 4 and Tunga penetrans (10), among others. It also occurs in parasitic nematodes such as 5 Onchocerca volvulus and an inflammatory reaction that can induce blindness (24, 22) 6 and has been implicated with Brugia malayi (36, 34). Wolbachia has recently been foun 7 in Angiostrongylus cantonensis, a nematode unrelated to filarias (37).

8 The sylvatic triatomine R. pallescens is considered the most important vector of the 9 trypanosomatids T. cruzi and T. rangeli in the neotropics. Its capacity to invade houses 10 situated near its natural habitat, the royal palm tree (Attalea butyracea) and to transmit T.

11 cruzi and Chagas' disease to humans has been well documented in Panama (23, 4, 32).

12 Wolbachia has previously been reported in only one individual of R. pallescens published 13 in a list of Panamanian species (42). However, information on the occurrence of the 14 bacterium among its population and the organ distribution in the host is not available. In 15 this work we also analyze if there is any correlation of the endosymbiont with the 16 presence of the parasites T. cruzi and T. rangeli.

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18 In this study we examined a total of 72 triatomine individuals of R. pallescens, 27 19 collected from their natural habitat and 45 from an insectarium. Wild specimens were 20 collected in regions of the Republic of Panama where Chagas' disease is endemic (Table 21 1). Insectarium specimens were obtained from the Instituto Conmemorativo Gorgas de 22 Estudios de la Salud (ICGES) and from the Centro de Investigaciones Parasitarias de la 23 Universidad de Panama (CIDEP).

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1 Each specimen was dissected and gonads, salivary glands, and intestines were extracted 2 under sterile conditions. The posterior intestine, rectal ampolla and salivary glands from 3 wild triatomines were homogenized and any trypanosomes present were observed under 4 the microscope or cultured in Grace medium to facilitate detection of T. cruzi and T.

5 rangeli (20, 21). Also, detection of T. cruzi and T. rangeli was carried out as well by 6 PCR, as previously described (45, 46, 7).

7 The presence of Wolbachia was detected in each organ by PCR using specific primers 8 16S rRNA and wsp genes as previously reported (41, 44). Standard reactions contained 9 0.5 jul of the template DNA (extracted with QIAGEN DNeasy tissue kit) in a final 10 volume of 10 jul plus 0.08 jul of dNTP's (25 mM) and 0.5 jul of the forward and the 11 reverse primers (10 p,M), 0.1 jul of Taq polymerase (5U/L), 0.5 pi of MgCl2 (50mM) and 12 0.4plofDMSO(5%).

13

14 The integrity of the total DNA extracted was verified by amplification of the 28S rRNA 15 gens as previously described (5), and DNA from Nasonia, positive and negative for 16 Wolbachia (kindly provided by Dr. Werren) was used as controls.

17 The results of the screening of the 27 wild triatomines are shown in Table 1. The study 18 revealed an infection with T. cruzi in 56% and with T. rangeli in 25% of the cases.

19 Simultaneous infection with T. cruzi and T. rangeli was also detected in 12% of the 20 specimens. The presence of T. cruzi in the wild insect is indicative of the elevated risk of 21 Chagas' disease infection in humans in the insect capture areas.

22

I *

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ie possibility of false

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(Table 2). Of those 1 PCR analysis with probes for the wsp gene detected the presence of Wolbachia in the 2 gonads and salivary glands of 100% of the insects while with the specific primers for 16S 3 rRNA detected Wolbachia in 95.9% of the cases. As recommended by Duron and 4 Gavotte (2007) (8), the two pairs of primers were used to rule out the possibility of false 5 negatives.

6 The analysis of specimens from the insectarium showed a very differe

7 51.0% of the insects were positive for Wolbachia with both probes (Table 2).

8 positive, 51.0% registered positive in the gonads, 44.4% in the salivary gland, and 94.0%

9 in the intestine.

10 Given Wolbachia's presence in all wild triatomines collected, it seems that the presence 11 of the endosvmbiont does not influence the susceptibility of the triatomines to become 12 infected by the parasitic protozoan T. cruzi, the etiological agent for Chagas' disease, and 13 T. rangeli, for which these insects are natural hosts.

14

15 Wolbachia is vertically transmitted by oocyst infection maintaining in this way a high 16 incidence in arthropods (33). Its presence has been reported in other tissues, even in nerve 17 tissue or in hemocytes (25). In order to determine the degree of infection by Wolbachia in 18 other organs of R. pallescens, apart from those used in the screening, an insectarium 19 specimen of R. pallescens was dissected to extract the hemolymph, the musculature, the 20 Malpighian tubules and intestine. Each organ was tested for the presence of Wolbachia 21 with specific primers for 16S rRNA and wsp genes. All PCRs were positive indicating 22 the bacterium was distributed throughout the tissues of the insect, and it is not restricted 23 to the digestive tract and gonads.

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1 The presence of Wolbachia both in the salivary glands as well as in the intestine might 2 be explained by the coprophagous and cannibalistic habits of these insects in the early 3 phases of their development, when they provide themselves with the symbionts essential 4 for their development (30, 31). This could also constitute the transmission and spreadin 5 mechanism of the endosymbiont among triatomines. To verify whether Wolbachia 6 present in the digestive products of the triatomine, feces were collected after feedin 7 insectarium specimen and probed with 16S rRNA and wsp gene primers. Both

>e dete

8 amplifications were positive. The fact that Wolbachia can be detected in the feces of 9 triatomines makes it unnecessary to neutralize the insect to determine the presence of this 10 endosymbiont, which represents an important methodological advantage.

11

12 In order to characterize the Wolbachia strain present in R. pallescens several PCR 13 products were sequenced. The sequence of the 16S rRNA gene obtained from feces of an 14 insectarium specimen was 99-100% identical to a Wolbachia endosymbiont of 15 Pseudolynchia canariensis (Ac. No. DQ115537), and other unculturable bacteria from 16 insects such as the cat flea Ctenocephalides felis (Ac. No. EF121347), and the ant lion 17 Myrmeleon mobilis (Ac. No. EF121347, DQ068883, DQ068882), and the fruit fly 18 Drosophila melanogaster (Ac. No. DQ981371, DQ981358, DQ981347). The same 16S 19 rRNA sequence was found in the feces of a wild Rhodnius collected in Chuzo (denoted 20 (e) in Table 1). The wsp gene sequence from this wild individual was compared with 21 those in the database constructed and maintained by Jolley and Baldo {Wolbachia MLST 22 Databases, (http://pubmlst.org/wolbachia/). The result of the comparison showed a 23 likeness of 96.10% with the allele 92 of the database. Given that the authors consider a

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1 single difference in the nucleotide sequence as different alleles, it would appear that the 2 strain of Wolbachia present in the triatomines is a novel strain and is not included in this 3 database.

4

5 The fbpA gene was also amplified and sequenced (15) from salivary glands of 9 6 specimens positive for Wolbachia (denoted (d) in Table 1). Direct observation

7 DNA chromatograms showed superimposed peaks at 16 different positions. These results 8 were interpreted as R. pallescens being infected by at least two Wolbachia strains.

9 Surprisingly, an analysis of the sequence of the fbpA gene from 3 insectarium specimens 10 (denoted (d) in Table 2) showed only 4 superimposed peaks. Although insectarium and 11 wild triatomines were collected from different areas of Panama, superimposed peaks 12 were found at the same positions in both groups. A possible explanation for this 13 observation is that both insect groups are infected by the same Wolbahcia strains and at 14 least one of those strains has been cured when triatomines are raised in laboratory 15 colonies. This result strongly indicates that the vertical transmission of Wolbachia could 16 be affected in insects raised under laboratory conditions. Some factors have previously 17 been described (33) to alter the presence and spread of Wolbachia in the insect. In our 18 case the exposure to high temperatures, the immune response of the vertebrate used for 19 laboratory feeding or genetic factors of the host could be relevant factors. It would be 20 interesting to study by means of which mechanisms triatomines, on being extracted from 21 their natural habitat and raised in laboratories, alter the development or spread of this 22 endosymbiont.

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1 The study of the presence of Wolbachia in triatomines opens up a new research area of 2 study into the possibility of using this endosymbiont to manipulate the reproduction of 3 these insects responsible for the vectorial transmission of Chagas' disease.

4

5 Our thanks go to Dr. J. Werren from the University of Rochester for his advice^H 6 Spanish Agency for International Co-operation to cover the travel expenses of 7 research team (A/5115/06 and A/8187/07), and to ICGES and CIDEP for providing

rom the

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8 insectarium triatomines. Dr. Susana Vilchez received a grant from the Pro grama Ramon y 9 Cajal (MEC, Spain and EDRF, European Union). We also thank Dr. J. Trout for revising 10 the manuscript.

11 12 13 14

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1 Table 1. The occurrence of trypanosomes and Wolbachia in R. pallescens collected from 2 several Panamanian districts. Males (M), females (F), and nymphs (N) were assayed for the presence of trypanosomatids and Wolbachia by PCR (a), microscopic examination (b) 4 or parasite isolation in mice (c) and recorded as positive (+) or negative (-) for those 5 organisms in the table. Not determined samples (ND) are also indicated. Samples used 6 amplifying and sequencing fbpA and wsp are denoted (d) and (e), respectively.

Species Sex

Trypanosome Wolbachia

T. cruzi

T. rangeli (a)

Gonads (a)

Salivary^

glands (a)

Region District

Rhodnius pallescens N + (a) - + ND Viento Fronto Chilibre

Rhodnius pallescens M + (a) + + + Viento Fronto Chilibre

Rhodnius pallescens M - (a) 1 + + + Viento Fronto Chilibre

Rhodnius pallescens M + (a) + + (d) + Viento Fronto Chilibre

Rhodnius pallescens M - (a)

K-

+ (d) + Viento Fronto Chilibre

Rhodnius pallescens F + (a) ^^^^^ + (d) + Viento Fronto Chilibre

Rhodnius pallescens M + (a) 1 1 ^^^^ ₊ ₊ Viento Fronto Chilibre

Rhodnius pallescens M + (a) - + + Viento Fronto Chilibre

Rhodnius pallescens F + (a) - + + Viento Fronto Chilibre

Rhodnius pallescens M - (a) - + + Viento Fronto Chilibre

Rhodnius pallescens M - (a) - + (d) + Viento Fronto Chilibre

Rhodnius pallescens M - (a) - + + Viento Fronto Chilibre

Rhodnius pallescens F + (b) - + + Loma Bonita Arraijan

Rhodnius pallescens F - (b) - + (d) + Santa Clara Arraijan

Rhodnius pallescens M + (b) + + (d) + Santa Clara Arraijan

Rhodnius pallescens M - (b) - + (d) + Santa Clara Arraijan

Rhodnius pallescens M - (b) + + (d) - Santa Clara Arraijan

Rhodnius pallescens F - (b) - + + Loma del Rio Arraijan

Rhodnius pallescens F - (b) + + + Santa Clara Arraijan

Rhodnius pallescens ND + (b)(c) ND + ND Carriazo Chepo

Rhodnius pallescens ND + (b)(c) ND + ND Playa Larga Chepigana

Rhodnius pallescens ND + (b) ND +(e) ND Chuzo Chepigana

n=27 M:17

F:6 N: 1 NO: 3

Pos.:15 Neg: 12

Pos.: 6 Neg: 18

ND:3

Pos.:27

Pos: 22 Neg: 1

ND:4

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1 Table 2. The occurrence of Wolbachia in insectarium specimens of R. pallescens. Male 2 (M), female (F), and nymph (N) were assayed for the presence of Wolbachia by PCR, 3 and recorded as positive (+) or negative (-) for this endosymbiont. Not determined 4 samples (ND) are also indicated. Samples used for amplifying and sequencing fbpA are 5 denoted (d).

Specie Sex

Wolbachia

Generation | Gonads Salivary

glands

Intestine

Rhodnius pallescens F ⁺ ⁺ ⁺ ND

Rhodnius pallescens F + + + ND

Rhodnius pallescens F + _ ₊ ND

Rhodnius pallescens M + + + ND

Rhodnius pallescen^^ F " ^ND 4°

Rhodnius pallescens F _ - ND 4°

Rhodnius pallescens M + + + 5°

Rhodnius pallescens M - - ^ND 6°

Rhodnius pallescens F - - ^ND 6°

Rhodnius pallescens M - - ND 4°

Rhodnius pallescens F + + + 5°

Rhodnius pallescens F + + - 5°

Rhodnius pallescens F - - ND 4°

Rhodnius pallescens N - - ND 4°

Rhodnius pallescens F ⁺ ⁺ + 6°

f

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Rhodnius pallescens F + + ND 3°

Rhodnius pallescens F + - + 3°

Rhodnius pallescens F + + ND ND

Rhodnius pallescens M + ND ND 3°

Rhodnius pallescens M + (d) ND ND 3°

Rhodnius pallescens F - ND ND 3°

Rhodnius pallescens M - ND ND 3°

Rhodnius pallescens F + (d) ND ND 4°

Rhodnius pallescens F + ND ND 4°

Rhodnius pallescens M - ND ND 4°

Rhodnius pallescens M + (d) ND ND 3°

n=45

F:29 M:14 N:2

+:23 -:22

+:16 -:20 ND:9

+:15 -: 1 ND:29

\

19