J Arthropod-Borne Dis, December 2017, 11(4): 446–452 O Páez-Rondón et al.: Revalidation of … 446 http://jad.tums.ac.ir Published Online: December 30, 2017 Original Article Revalidation of Synonymy between Nesotriatoma flavida and N. bruneri (Hemiptera, Reduviidae, Triatominae) Oscar Páez-Rondón 1,2, Fernando Otálora-Luna 2, *Elis Aldana 3 1Unidad de Articulación Comunitaria, Centro Multidisciplinario de Ciencias, Instituto Venezolano de Investigaciones Científicas, Mérida, República Bolivariana de Venezuela 2Laboratorio de Ecología Sensorial, Centro Multidisciplinario de Ciencias, Instituto Venezolano de Investigaciones Científicas, Mérida, República Bolivariana de Venezuela 3Laboratorio de Entomología “Herman Lent”, Departamento de Biología, Facultad de Ciencias, Universidad de Los Andes, Mérida, República Bolivariana de Venezuela (Received 6 June 2016; accepted 17 Oct 2017) Abstract Background: We analyzed the external morphology and the external male genitalia of specimens of Nesotriatoma flavida of a laboratory colony founded with specimens from Guanahacabiles in Pinar del Río Province in the west of Cuba in 1980. This species was at first identified as different from N. bruneri and then later considered synonymous. Methods: We proposed to revise the morphological characters with which these species were considered as different and then later synonymous, such as the fossula spongiosa or spongy grooves, length of the first antenna segment, relationship length of eye to interocular distance, the form of the phallosome, phallosome support, and the endosome processes. Results: The results of the analyses of these characters in the specimens of our colony, and of the corresponding works where the separation and synonymy of these species has been proposed, allow us to sustain the revalidation of the synonymy between N. flavida and N. bruneri. Conclusion: Based on the body external morphology and the male external genitalia of N. flavida it is concluded that N. flavida and N. bruneri are synonymous species. Keywords: Chagas’ disease, Phallosome, Morphology, Taxonomy Introduction A new genus was describes, Nesotriatoma, in which he includes Nesotriatoma flavida (initially described as Triatoma flavida) col- lected in the western region of the island of Cuba and also a new species Nesotriatoma bruneri, collected in the eastern region of this island (1). Nesotriatoma flavida has a short first segment of the antenna that does not reach the peak of the clypeus, well developed spongy grooves on the front legs of the females, and eyes as wide as a third of the interocular dis- tance. Nesotriatoma bruneri describes as hav- ing a long first antenna segment, reaching or passing the peak of the clypeus, no spongy grooves on the front legs of the females, and the width of the eyes as more than half the in- terocular distance. However, later proposes synonymy between N. bruneri and N. flavida, given the plasticity of the characters with which was diagnosed both species (2). Some authors (3) propose as not valid the synonymy proposed earlier (2), based on mor- phological differences in the external geni- talia of the male and include both species in the Triatoma genus, already considered as synony- mous with Nesotriatoma in (2-4). The sepa- ration of both species and their inclusion are accepted in the Triatoma genus (5). Posteriorly, the separation of the two spe- cies is maintained but based on phylogenetic analyzes include them in the Nesotriatoma genus (6). In a checklist of the current valid *Corresponding author: Dr Elis Aldana, E-mail: elis.aldana@gmail.com, aldana@ula.ve J Arthropod-Borne Dis, December 2017, 11(4): 446–452 O Páez-Rondón et al.: Revalidation of … 447 http://jad.tums.ac.ir Published Online: December 30, 2017 species of the subfamily Triatominae also con- sider valid Nesotriatoma genus (7). This genre is composed by the species N. flavida, N. bruneri and N. obscura (flavida complex). However, recently (8) by genetic analyzes pub- lished an article questioning the specific status of N. bruneri because this specie presented the same cytogenetic characteristics of N. flavida and an extremely low genetic distance (0.004) (8). The specimens with proposed the separa- tion of N. flavida and N. bruneri based on the morphological characters of the external genitalia of the males insects were sent by Dr W Torrealba (3). “Dr Torrealba sent us 5 specimens from the corresponding group. One specimen was identified as a typical T. flavida female captured in a human dwelling, and 4 specimens (two males and two females) captured in an animal burrow (?) of Pinar del Río Province, Guanahacabiles in the west of Cuba on 4-11-1979”. With individuals from the same locality and captured on the same date a colony was established at the Universidad de La Habana, Cuba and with individuals of this colony was founded the colony of N. flavida at our la- boratory in Mérida, Venezuela whose mem- bers were used in this work for analyses of external morphological characteristics and of the male external genitalia, with the aim of verifying whether the triatomines that form the colony were N. flavida or N. bruneri, in view of the polemic in the taxonomic status of these species. Materials and Methods Entomologic material Nine males and nine females of N. flavida from the Laboratory of Entomology “Herman Lent” (HLEL), Venezuela, were examined, being maintained at 28 °C, 50% relative hu- midity, and fed hen blood. This colony was founded on 18-6-1980 with eggs, 3 females, 2 males, and 5 fifth instar nymphs brought by Dr Scorza from the colony identified as Triatoma flavida in the Universidad de La Habana, Cuba. Morphological analyses The analysis of the external morphology of the adult body was carried out following the taxonomic keys (4), and the descriptions of both species (1, 5). The analysis of the mor- phology of the external male genitalia was done following the terminology employed and descriptions of the external male genitalia of both species (3-5). Both morphological studies were observed with a Leica M205A stereoscopic microscope, the images were taken with a CMOS camera (EOS Rebel T3, Canon). The genitalia were dissected at the level of the two final ab- dominal segments, and processed in the fol- lowing fashion: KOH (Eka, Chemicals) at 10% in a mortar and heated without boiling until soft, later treated according to treatment 1: 36h in phenol (90%, Honeywell Riedel-de- Haën) and 48h in guaiacol (Scharlau Chemi- cals, S.A.), or treatment 2: 72h both in the phenol and in the guaiacol. Finally, they were mounted in DPX (Industrias Químicas ERBA, C.A.) on a microscope slide and observed un- der a stereo microscope (Leica M205). The pictures were taken with a CMOS camera (Leica EC3) coupled to this microscope. The drawings of the borders both of the silhou- ette of the phallosome as well as its support in the male external genitalia were done us- ing a raster graphics editor (Photoshop CS6, 13.0 x64). Results External morphology of the body General color: clear brown. Body and co- rium are practically hairless, hairs scarce and very short. Femur: variable: from completely dark or completely light, or the proximal half dark and the distal light, with the presence or absence of denticles on the front legs. Tibias J Arthropod-Borne Dis, December 2017, 11(4): 446–452 O Páez-Rondón et al.: Revalidation of … 448 http://jad.tums.ac.ir Published Online: December 30, 2017 and distal parts of the end of the femur have a light color (Figs. 1, 2). Spongy grooves under- developed, present on the front legs of both males and females (Fig. 3). Pronotum is dark, except for the humerus, anterolateral angles, and the submedian carinae, which are of a yellow color. Pronotum has prominent anter- olateral and humeral angles, anterior lobes with very noticeable lateral and discal tubercles and with yellow-edged halos, a butterfly figure at the union of the two lobes. On the front edge of the scutellum disk, 1+1 prominent tuber- cles pointed forwards and touching the back edge of the pronotum. Median region of the scutellum has noticeable transverse grooves (Fig. 4). Hemelytron is covering laterally part of the tergites and reaching the seventh urotergite, extensively spotted towards the back part and clearly defined dark stains on the front portion (Fig. 5). Proportion eye width: interocular distance 1:2 (Fig. 6). Third rostrum segment is shorter than the others. First antenna segment does not overshoot the peak of the clypeus (Fig. 7). Morphology of the male external genitalia Phallosome lengthy, oval, and with con- cave base when the genitalia are maintained 36h in phenol and 48h in guaiacol (Figs. 8, 9, 10(3)). Phallosome lengthy, hexagonal, and concave base when maintained 72h in both phenol and guaiacol (Figs. 11, 12, 13(3)). The process of the endosome extends approximately 75% of its length from the base of the sup- port of the phallosome, with a lengthy, very wrinkled and grooved appearance when the genitalia are maintained 36h in phenol and 48h in guaiacol (Fig. 8) as well as when main- tained 72h in both the phenol and the guaia- col (Fig. 11). The phallosome support long, with cylindrical base, side edges converging towards the outer end of the base under ei- ther of the treatments of the genitalia (Figs. 14, 15(3)). Median process of pygophore sim- ple and pointed (Fig. 16). Table 1. Comparison of external morphological characters of the body and of the external male genitalia of Nesotri- atoma flavida and Nesotriatoma bruneri Character (1) N. flavida (4) (3) N. flavida Present work N. flavida N. bruneri N. flavida N. bruneri First antenna segment size Sh L Sh Sh Sh Sh Spongy groove distribution F:An F:An M:An, Md; F:V M:An, Md; F:V M:An, Md; F:An, Md M:An; F:An Spongy groove degree of de- velopment D N/D N/D N/D SD SD Ratio eye width: interocular distance 1:3 1:2 1:3 1:2 1:2 1:2 General body color Brown and ochre Mottled brown Light hazel Light brown Dark mottled hazel Light hazel Form of distal protuberances of anterior pronotal lobe Sm, Rd, Prm Prm Rd, Subc N/D N/D Prm Form of lateral protuberances of anterior pronotal lobe Sm, Rd, Prm Prm Rd, Subc N/D N/D Prm Presence of denticles in anterior and median femurs A P P P P V Form of phallosome N/D N/D N/D L, Hx, BsCc L, Ov, BsCc tr1:L, Ov, BsCc tr2:L, Hx, BsCc J Arthropod-Borne Dis, December 2017, 11(4): 446–452 O Páez-Rondón et al.: Revalidation of … 449 http://jad.tums.ac.ir Published Online: December 30, 2017 Form of phallosome support N/D N/D N/D L, BsCy, BrLPr L, BsCy, BrLCv tr1:L, BsCy, CvLE tr2:L, BsCy, CvLE Form of endosome processes N/D N/D N/D L, Al Sh, W L, W Median processes of pygophore N/D N/D N/D N/D N/D S, Pn Sh: short, L: long, F: female, M: male, An: anterior, Md: median, V: variable, D: developed, N/D: not described, SD: slightly developed, Sm: small, Rd: round; Prm: prominent, Subc: subconic, A: absent, P: present; Hx: hexagonal, BsCc: concave base, Ov: oval, BsCy: cylindrical base, PrLE: parallel lateral edges, CvLE: convergent lateral edges, Al: aliform, W: wrinkled, S: simple, Pn: pointed, Sth: smooth, tr1: treatment 1, tr2: treatment 2. Numbers in parenthesis correspond to citations in References. Figs. 1–7. External morphology of the body of Nesotriatoma. flavida. 1: Dorsal view of complete male body. 2: Femur and tibia of the female. 3: Spon- gy groove on the distal end of the foreleg tibia. 4: Dorsal view of the pronotum and scutellum of the female. 5: Dorsal view of the scutellum and hemely- trons of the male. 6: Dorsal view of the male head. 7: Ventral view of male head Figs. 8–16. Morphology of external male genitalia. 8: Ventral view of phallosome, treatment 1 (see Mate- rials and Methods). 9: Phallosome silhouette, treat- ment 1. 10: Phallosome silhouette of N. bruneri ac- cording to (3). 11: Ventral view of phallosome, treat- ment 2 (see Materials and Methods). 12: Phallosome silhouette, treatment 2. 13: Phallosome silhouette of N. flavida according to (3). 14: Phallosome support, a: treatment 1, b: treatment 2. 15: Phallosome support, a: N. flavida, b: N. bruneri, according to (3). 16: Median processes of pygophore. Ph: phallosome, PrEn: endo- some process, SPh: phallosome support, PrP: median process of pygophore. Discussion The characters: length of the first antenna segment in relation to the clypeus, presence of spongy grooves, and ratio eye breadth to interocular distance as features that distinguish N. flavida from the new species he calls N. bruneri (1), all of which later (2) considers so variable that they do not justify the distinc- tion between the species, and so he considers them synonymous. The external male genita- lia of N. flavida was described as a quadran- gular phallosome, phallosome support with par- allel edges, semicircular vesica, and long en- dosome processes, while those of N. bruneri is described as being an oval phallosome, con- vergent edges of the phallosome support, ovoid- al vesica, and short endosome processes (3). Examining the illustration was provided (Fig. 13), the phallosome of N. flavida was not quadrangular, but rather hexagonal (Figs. 11, 12). The endosome process extends from the middle part of the phallosome to the vesica in N. flavida, and indicated it in N. bruneri, but it is indistinguishable in such illustration (not showed). Although we observed that the phallosome support is pointed in both spec- imens: N. flavida described in our work (Figs. 14a,b) and in N. bruneri described, according to these authors the phallosome support is blunt in N. flavida and more pointed in N. bruneri (Fig. 15a,b) (3). Although we observed spon- gy grooves in female and males, the presence of spongy grooves varies according to whether the legs are frontal or medial and according to sex, the authors did not describe this structure Table 1. Continued… J Arthropod-Borne Dis, December 2017, 11(4): 446–452 O Páez-Rondón et al.: Revalidation of … 450 http://jad.tums.ac.ir Published Online: December 30, 2017 in N. bruneri. They described the interocular distance as being double the eye width in both species (3). Finally, we observed that the first antenna segment does not reach the peak of the clypeus in any of the species (Fig. 1). In our opinion the distinction between N. flavida and N. bruneri proposed (3) is poorly based, the description of the phallosome sil- houette is erroneous, the imprecise illustration and distinction of the vesica as semicircular in N. flavida and ovoidal in N. bruneri, the poor illustration of the endosome process in N. bruneri, the imprecision with which the en- dosome process is considered “large”, and the absence in that paper of a comparative analy- sis between the species based on those char- acters that first distinguished the species and then later considered them synonymous given the phenotypical plasticity of the length of the first antenna segment with respect to the clyp- eus, the spongy grooves, and the relation be- tween eye width and interocular distance (1, 2). In the present work it was found that the specimens identified as N. flavida selected from the colony in our laboratory, formed from the same group of insects collected in Guanahacabiles in Pinar del Río Province in the west of Cuba (3), present the following features: 1) the first antenna segment does not overshoot the clypeus peak like that de- scribed in N. flavida, and in N. flavida and N. bruneri (1,3), 2) spongy grooves poorly de- veloped, present in the forelegs of both males and females, as describe for N. bruneri (3), and differing from what observes as well de- veloped grooves in the female and absent from the males of N. flavida, and missing in both sexes of N. bruneri, (1) and also differing from what observe in finding the grooves present on the front and middle legs of the males and variable presence on the forelegs of the N. flavida female (3), 3) ratio eye width–inter- ocular distance 1:2, similar to what describes for N. bruneri, for N. flavida and N. bruneri, and differing from the description in for N. flavida (1:3) (1,3), 4) when the genitalia were treated 36h in phenol and 48h in guaiacol, the phallosome silhouette is found to be oval, similar to that described for N. bruneri (3), (Fig. 10), while on the other hand, phallosome has a hexagonal silhouette when treated 72h in phenol and 72h in guayacol, similar to that described as “quadrangular” in N. flavida (Fig. 13), 5) phallosome support base cylindrical, pointed peak and convergent edges irrespec- tive of the phenol and guaiacol treatments, similar to the cylindrical support base de- scribed for N. flavida (3), (Fig. 15a), and to the pointed peak and convergent edges of N. brunei by these same authors (Fig. 15b), and 6) endosome processes long and wrinkled in either of the different times in phenol and guaiacol, for N. bruneri (3) (Table 1). In the present work, differences were found in char- acteristics of the external male genitalia ac- cording to as to how they were treated in phenol and guaiacol, which exemplifies the im- portance of a detailed description of the treat- ment protocols of the genitalia, an aspect not treated (3). Our proposal for the revalidation of the synonymy between N. flavida and N. bruneri is based on the following facts: 1) according to our results and the corresponding ones (1, 3), the morphological characteristics such as length of the first antenna segment, presence of spongy grooves, ratio eye width: interocu- lar distance, general body color, and denticles of femur are quite variable, 2) in our results the form of the male external genitalia varies according to the hours of treatment in phenol and guayacol, showing characters like either what described for N. flavida or for N. bruneri (3). Based on the sequence of the gene 16S rRNA, was found differences (ca. 1.37%) be- tween N. flavida and N. bruneri (6). Although there is no agreement about the amount of dif- ference at the sequence level that would con- stitute a proof of distinction between species, the differences at the sequence level of the 16S rRNA gene (not a protein coding gene but an RNA structural gene with a slow rate of change), would not alone permit sustaining the J Arthropod-Borne Dis, December 2017, 11(4): 446–452 O Páez-Rondón et al.: Revalidation of … 451 http://jad.tums.ac.ir Published Online: December 30, 2017 thesis of different species. This kind of evi- dence is valid if it lends support to other char- acters like morphological, ecological, behavior- al, etc. Since there is no evidence of these dif- ferences, the distinction based solely on the 16S rRNA gene sequence (6) is for the moment in- sufficient to classify N. flavida and N. bruneri as different species. The genetic variability of N. flavida and N. bruneri were analyzed by means of the RAPD-PCR technique and found differences between the species (9). This technique is not valid to separate species, but rather is used for polymorphism analysis or intraspecific vari- ation, and so found does not invalidate the syn- onymy between these two species. By means of an antenna phenotype discrim- inating analysis manage to separate N. flavida from N. bruneri (10), but since they do not find differences upon comparing the same sex between the two species, the separation should not be taken as definitive, but rather subject to later molecular, ecological, and morpholog- ical analyses to clarify the taxonomic status of both species. Given the ambiguity of the differences found with the discriminating anal- ysis of the antenna phenotype reported by these authors, we also consider that their re- sults do not constitute evidence that invali- dates the synonymy between N. flavida and N. bruneri. Based on the foregoing exposition, we con- sider that maintaining the synonymy between N. flavida and N. bruneri is more solidly jus- tified than considering them different species. The synonymization of N. bruneri with N. flavida is of utmost importance to public health in Cuba because the country ceases to have four species of triatomine [Bolbodera scabrosa, N. bruneri, N. flavida, T. rubrofasciata] and has only three species now, being N. flavida with wide geographic distribution. Furthermore, the synonymization allows clarifying evolu- tionary questions, as for example the occupa- tion of the Antilles by this species associated with a species of rodent about 14.8 to 18.8 Ma (7, 11). Conclusion Based on the body external morphology and the male external genitalia of N. flavida it is concluded that N. flavida and N. bruneri are synonymous species. Acknowledgements To CDCHTA-ULA project C-1932-15- 03-A. The authors declare that there is no conflict of interest. References 1. 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