Bioacoustics of Triatoma bahiensis

Diagnosis. Triatoma bahiensis ( Figs. 1 , 3A –B) is black with orange-red spots on the connexivum that are greater than those of T. lenti ( Figs. 1 , 3 C–D). The hemyelitrum of T. bahiensis presents discoidal cells black, does not reach the apex of the abdomen and possesses two orange-red spots on each side of the corium ( Figs. 1 , 3A –B), whereas the hemyelitrum of T. lenti is totally black without spots in the corium. Morphological analysis. The exochorial cells of T. bahiensis eggs ( Fig. 4 A) revealed a higher quantity of pores than those of T. lenti ( Fig. 4 B). These pores are concentrated in the central region of T. bahiensis egg cells ( Fig. 4 A), while in T. lenti the pores are uniformly distributed and also found on egg cell borders ( Fig. 4 B). The posterior portion of the central depression of the scutellum is rounded in T. bahiensis ( Fig. 4 C) and tapered in T. lenti ( Fig. 4 D). The first abdominal segment has two lateral prominences in T. bahiensis ( Fig. 4 C). These prominences are absent in T. lenti ( Fig. 4 D). Morphological analysis of the ventral thorax showed that the anterior region of the prothorax (near the stridulatory sulcus) in T. lenti presents a depression ( Fig. 5 D), which is absent in T. bahiensis ( Fig. 5 A). Furthermore, the stridulatory sulcus of T. bahiensis ( Fig. 5 A) is narrower than that of T. lenti ( Fig. 5 D). In T. bahiensis , the posterior region of the stridulatory sulcus has rounded and well-defined edges ( Fig. 5 A), whereas in T. lenti this region has rough edges ( Fig. 5 D). The mesothorax of T. bahiensis has a central longitudinal projection that is rectangular in shape ( Fig. 5 B). In T. lenti , however, the mesothorax is smooth and rounded ( Fig. 5 E). No distinctive features were observed between the metathoraces of T. bahiensis and of T. lenti ( Figs. 5 C, F). Morphometric analysis. All of the morphometric characteristics analyzed in males and females of T. bahiensis were smaller than those of T. lenti , except for the third segment of the proboscis, which is the same size in females of both species and larger in T. bahiensis males ( Tables 1 and 2 ). Triatoma lenti Triatoma bahiensis Min Max X SD S2 Min Max X SD S2 Length of head* 4.83 5.48 5.30 0.23 0.05 4.70 5.14 4.98 0.12 0.01 Length of thorax* 5.79 6.80 6.33 0.31 0.01 5.00 5.86 5.48 0.33 0.11 Length of abdomen* 13.13 15.01 13.94 0.65 0.42 11.09 13.06 12.40 0.62 0.38 Length of 1st rostral segment 1.47 1.82 1.61 0.11 0.01 1.38 1.75 1.53 0.10 0.01 Length of 2nd rostral segment* 2.73 3.12 2.95 0.12 0.01 2.50 2.93 2.74 0.15 0.02 Length of 3rd rostral segment 1.20 1.31 1.24 0.03 0.00 1.17 1.35 1.24 0.06 0.00 Inner distance between eyes 1.33 1.43 1.37 0.03 0.00 1.27 1.44 1.34 0.05 0.00 Outer distance between eyes* 2.34 2.61 2.48 0.09 0.01 2.15 2.51 2.38 0.10 0.01 Length of eggshells* 2.27 2.73 2.50 0.11 0.01 1.88 2.17 2.04 0.08 0.00 Diameter of the opercular opening* 0.72 0.82 0.76 0.03 0.01 0.62 0.78 0.71 0.03 0.00 Area of opercular opening* 0.40 0.52 0.46 0.03 0.01 0.30 0.47 0.40 0.04 0.00 *differences between species are significant (analysis of variance—p <0.05); Max: maximum; Min: minimum; SD: standard deviation; S2: variance; X: average. Triatoma lenti Triatoma bahiensis *differences between species are significant (analysis of variance—p <0.05); Max: maximum; Min: minimum; SD: standard deviation; S2: variance; X: average Min Max X SD S2 Min Max X SD S2 Length of head* 4.56 5.31 5.12 0.19 0.04 4.54 5.07 4.85 0.16 0.03 Length of thorax 5.73 6.20 5.94 0.19 0.02 5.24 6.19 5.84 0.31 0.09 Length of abdomen* 11.89 14.27 13.61 0.63 0.36 11.44 13.30 12.64 0.55 0.30 Length of 1st rostral segment* 1.39 1.73 1.59 0.08 0.00 1.42 1.66 1.52 0.08 0.01 Length of 2nd rostral segment 2.61 3.02 2.85 0.13 0.02 2.53 2.93 2.78 0.13 0.02 Length of 3rd rostral segment* 1.10 1.27 1.19 0.05 0.00 1.18 1.33 1.26 0.05 0.00 Inner distance between eyes 1.17 1.34 1.25 0.04 0.00 1.16 1.32 1.23 0.05 0.00 Outer distance between eyes 2.23 2.53 2.40 0.08 0.00 2.23 2.50 2.35 0.09 0.01 Wings of T. bahiensis were smaller than those of T. lenti (p<0.01). The first two discriminant factors explained 76% and 21% of wing shape variation, respectively. Triatoma lenti and T. bahiensis were well separated in discriminant function analysis plots, and wing shape differences between species were evident, T. lenti wings being narrower than in T. bahiensis ( Fig. 6 ). No allometric effects were observed on the regression analyses between shape variables and size of wings (R2 = 0.01, p = 0.94). Experimental crosses. Triatoma bahiensis female × T. lenti male: out of 5 couples crossed, four produced fertile eggs and only 4% reached adulthood ( Table 3 ). The F 1 egg hatching rate was 81.3%, but the coupling did not produce adults (F2 generation). Generation T. bahiensis female x T. lenti male T. lenti female x T. bahiensis Nº of eggs laid male Nº of eggs hatched (%) Nº of adults (%) Nº of eggs Nº of eggs laid hatched (%) Nº of adults (%) F1 271 70 (25.8) 11 (4) 239 84 (35.1) 2 (0.8) F2 107 87 (81.3) - - - - Triatoma lenti female × T. bahiensis male: only one of the five couples produced fertile eggs; only two eggs reached adulthood ( Table 3 ). No eggs were generated from this offspring. Despite the low egg viability in both experimental crosses, no abnormalities were found in the hybrids. Cytogenetic analysis. The analysis of the early first meiotic prophase I of T. bahiensis ( Fig. 7 A) revealed a large chromocenter made up of the association of both sex chromosomes plus two autosomal pairs (arrow) and many heterochromatic blocks arranged inside the nucleus (arrowheads). In the diplotene stage ( Fig. 7 B), T. bahiensis presented an association between two autosomal bivalents (arrowheads) with sex chromosomes (arrow). In diakinesis or in later diplotene ( Fig. 7 C), this species presented ten autosomal bivalents and two (XY) sex chromosomes with heterochromatic blocks in one or both chromosomal ends of the autosomes. Note that the Y sex chromosome (arrow) is larger and more heterochromatic that the X chromosome. Phylogenetic analysis. The three specimens studied of each species did not present intraspecific variability. The comparison of Cyt b gene fragments between T. lenti and T. bahiensis revealed 17 variable sites, and pairwise sequence divergence was 2.5% ( Table 4 ). This value is higher than that found for T. b. brasiliensis and T. b. macromelasoma (2.0%), and it is close to the value found for the T. lenti and T. melanica (2.7%). Bayesian analysis grouped T. lenti , T. bahiensis , and T. melanica into a monophyletic clade ( Fig. 8 ). The other species of the T. brasiliensis complex are located in another clade. Phylogenetic analysis of T. lenti and T. bahiensis with T. melanica suggests that T. bahiensis and T. lenti represents two new members of the T. brasiliensis complex. Furthermore, the value of genetic distance between T. lenti and T. bahiensis support the revalidation of T. bahiensis . The sequence analysis of T. lenti and T. bahiensis with other species of the T. brasiliensis complex revealed 75 variable sites and three autapomorphies for T. bahiensis . All branches of the clade had 100% supports.