J Arthropod-Borne Dis, September 2018, 12(3): 262–268 E Franzim-Junior et al.: Biology of … 262 http://jad.tums.ac.ir Published Online: September 30, 2018 Original Article Biology of Meccus pallidipennis (Hemiptera: Reduviidae) to Other Conditions Than That Encountered in Their Native Habitat Edson Franzim-Junior 1, Maria Tays Mendes 1, Ana Carolina Borella Marfil Anhê 2, Thiago Alvares da Costa 1, Marcos Vinicius Silva 1, César Gómez Hernandez 1, Afonso Pelli 1, Helioswilton Sales-Campos 1, *Carlo Jose Freire Oliveira 1 1Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brazil 2Instituto de Tecnologia e Ciências Exatas, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brazil (Received 28 Sep 2016; accepted 17 July 2018) Abstract Background: Meccus pallidipennis (Hemiptera: Reduviidae) is only found in Mexico and is one of the most important vectors for Trypanosoma cruzi transmission there. Because data concerning the ability of this bug to adapt to different environments are scarce, we aimed to elucidate its biology, behavior and ability to acclimatize to different environ- mental conditions. Methods: From the eclosion of 90 1st instar nymphs, development was followed until the adult phase. Adults were fed after 30 days of fasting, and the average amount of blood ingested, the time between the beginning of the blood meal and the production of feces, and the frequency of stools/insect were recorded during their meals. After taking a blood meal, couples were isolated and monitored for 21 days, during which eggs were collected weekly. Results: The development of M. pallidipennis took 171.74±7.03 days to complete its life cycle, and females ingested larger amounts of blood than males. Oviposition was constant and did not demonstrate a significant decrease during this study. Conclusion: Meccus pallidipennis was able to acclimatize to fluctuating laboratorial conditions other than those nat- urally found in Mexico. Keywords: Behavior, Biology, Hemiptera, Meccus pallidipennis, Triatominae Introduction Chagas disease affects approximately 10 million people around the world, and at least 25 million are exposed to risk of infection (1). The protozoan Trypanosoma cruzi is the caus- ative agent, and the most frequent form of trans- mission is through the blood-sucking arthro- pods known as triatomines or “kissing bug” in- sects. After taking a blood meal, infected insects release feces contaminated with infectious meta- cyclic trypomastigotes adjacent to the site of the bite (2). Because insect vectors are the main route of transmission in endemic countries, the most effective strategy to restrain the spread of disease is to better understand the vector be- havior and biology. During their life cycle, triatomines go through five different nymphal stages before entering the adult phase when they reach their sexual maturity and are able to reproduce. Their biology is species-specific and depends on environmental conditions, such as temper- ature and humidity range (3). Additionally, as- pects concerning food intake may also have an impact on their epidemiology. In this con- text, there is a positive relationship between food intake and feces production, which to- gether with the times for feeding and evacua- tion, are key elements that dictate the vector efficiency and disease spread (4). *Corresponding author: Dr Carlo Jose Freire Oliveira, E-mail: carlo@icbn.uftm.edu.br http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 262–268 E Franzim-Junior et al.: Biology of … 263 http://jad.tums.ac.ir Published Online: September 30, 2018 So far, more than 140 species of triatom- ines have been identified. Among them, Meccus pallidipennis Stål (Hemiptera: Reduviidae), formerly known as Triatoma pallidipennis, is primarily found on the Pacific coast of Mex- ico and represents one of the main vectors of Chagas disease in that country (5). This insect is found either in sylvan or domestic reser- voirs (6). In urban areas, M. pallidipennis is associated with the presence of dogs, squir- rels, pigs and opossums near houses and un- constructed areas (7). The environment seems to affect some crucial aspects concerning the development, reproduc- tion and the potential of M. pallidipennis to transmit Chagas disease in Mexico (8). Because of the epidemiological importance of M. pallidipennis in Mexico and the rele- vance of this vector to different environments, we aimed to elucidate the biological and be- havioral aspects of this species under labora- torial fluctuating conditions of humidity and temperature. For this purpose, we investigated the biology and timing of the different stages of development, feeding behavior and oviposition. Materials and Methods Meccus pallidipennis Meccus pallidipenis specimens were origi- nated from the region of Cienega state of Ja- lisco, and gently donated by Dr José Alejandro Martínez Ibarra from Universidad de Guada- lajara, Mexico. Then, the second generation of triatomines were raised and kept in small plastic flasks (6.0cm height x 7.0cm diameter) until they reached the 4th nymphal instar at the insectary of the Federal University of Triângulo Mineiro (UFTM). Older nymphs and adults were kept in intermediate containers (7.5cm height x 11.0cm diameter). During the period of the study, triatomines were submitted to a natural light cycle and fluctuating environmen- tal conditions of temperature (max: 31.03±0.81 °C and min: 18.87±1.387 °C) and humidity (56.85±25.66%) in Uberaba, Minas Gerais, Brazil (19° 44′ 52″ S, 47° 55′ 55″ W–823m above sea level). Specimens were fed on im- mobilized and anesthetized Swiss mice week- ly. Mice were from the animal facility at the Parasitology Division of the UFTM in Uber- aba, Minas Gerais, Brazil and were anesthe- tized by using sodium tyopenthal (40.0mg/ kg intraperitoneal-i.p.). All procedures were submitted and ap- proved by the Institutional Animal Care and Use Committee of the Federal University of Triângulo Mineiro (Brazil) under protocol 307. Life cycle of Meccus pallidipenis We aimed to demonstrate the egg-to-adult developmental time under the aforementioned environmental conditions. After oviposition, eggs were collected and monitored until eclo- sion. Then, 90 nymphs were divided in colo- nies containing 30 insects each, and the time to reach the adult phase was recorded. Triatom- ines in the 1st nymphal stage were fed 10 days after eclosion (2nd and 4th nymphal stages) and were followed until the next molt. Insects in the 5th nymphal stage were fed twice, on the 10th and 20th days after molting. Only nymphs that fed were considered viable and able to develop from one developmental stage to an- other, and 5th instar nymphs were considered viable only after the second feeding. Feeding behavior analyses verified the av- erage time of feeding, amount of blood ingest- ed, and time between blood meal and excre- tion. During the blood meal, the following as- pects were recorded: feeding time, amount of blood ingested, time until first excretion and frequency of feces. The frequency of feces/ meal was also recorded. Randomly, 20 adult insects (10 males and 10 females), fasting for 28 days, were selected from pre-existing col- onies. Each triatomine was individually allo- cated to plastic receptacles and weighed be- fore and after the blood meal using an analyt- ic scale (UniBloc Shimad- zu AUY220, Kyoto, Japan). The blood ingestion rate was individ- ually calculated by dividing the amount of blood ingested by the feeding duration. The http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 262–268 E Franzim-Junior et al.: Biology of … 264 http://jad.tums.ac.ir Published Online: September 30, 2018 amount of feces/meal was calculated by divid- ing the frequency of total defecations per gen- der by the number of triatomines from each group/gender. Both the blood ingestion rate and the amount of feces per meal were calculated as previously describe (9), with some modifica- tions. Oviposition during different fasting times The oviposition was followed during dif- ferent fasting times to evaluate the impact of fasting on the production of eggs. Thus, 8 adult couples were randomly chosen, isolat- ed, and fed only once, and oviposition was monitored daily over 21 days. Eggs were collected on days 7, 14 and 21 after feeding and kept in different containers for analysis. Statistics For all variables, the normal distribution and homogeneous variance were tested. Ow- ing the size of our groups, we used non- parametric Mann-Whitney tests to compare time of feed- ing, blood ingestion and blood ingestion rate. To address the differences between the per-centage of males and fe- males producing feces a Fisher exact test was used. Since we had only eight couples, a non-parametric method (Friedman test) was used to compare oviposi- tion among weeks. The results were expressed as means±standard error of the means. The dif- ferences were considered statistically signifi- cant when P< 0.05 (5%). All analyses were per- formed using GraphPad Prism 5.0 soft- ware (San Diego, CA). Meccus pallidipennis developed under fluctuating environmental conditions To study the impact of fluctuating envi- ron-mental conditions on the development of M. pallidipennis, 90 nymphs were randomly se- lected, and their development was fol- lowed from hatching to adulthood. The mean time for eclosion was 20.15±0.74 days (Ta- ble 1). Furthermore, the number of days nec- essary to complete each instar increased when compared to the previous stage of de- velopment as follows: 22.13±0.83, 23.35±0.85, 25.13±1.03, 31.00±1.44 and 50.00±2.60 days, respectively, from 1st nymphal instar to adult (Table 1). Meccus pallidipennis females ingested larg- er amount of blood than males Because we observed that M. pallidipen- nis was able to properly develop under the local en- vironmental conditions, we next assessed their feeding behavior. The results showed that fe- males ingested larger amounts of blood than males (Table 2). No differences between gen- ders were observed for the feeding duration and blood ingestion rate (Table 2). Furthermore, 70% of adult males defecated in 26.69±7.98 minutes after starting the blood meal and had 0.80±0.63 feces/meal (Table 3), while females had 1.30±0.48 feces/meal during 25.43±7.83 minutes (Table 3). Though slightly different, no significant differences were observed be- tween the genders on these parameters. Oviposition Because triatomines were able to devel- op and fed under the conditions tested, we next investigated their ability to lay eggs. Overall, 140, 120 and 122 eggs were laid during the first, second and third week, re- spectively (Fig. 1a). Weekly, the oviposition rate was 17.50±4.81, 15.00±5.63 and 15.50±5.09, respectively, from the first to third week (Fig. 1b). Furthermore, daily ovi- position was recorded, and the rates were 2.50±0.68, 2.14±0.80 and 2.21±0.72, re- spectively, for the first, second and third week (Fig.1c). However, differences among the weeks were not significant. Table 1. Time of transition between developmental stages http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 262–268 E Franzim-Junior et al.: Biology of … 265 http://jad.tums.ac.ir Published Online: September 30, 2018 Developmental stage Median±SD (days) Eggs - N1 (n=80) 20.15±0.74 N1-N2 (n=56) 22.13±0.83 N2-N3 (n=49) 23.35 ±0.85 N3-N4 (n=39) 25.13 ±1.03 N4-N5 (n=27) 31.00 ±1.44 N5–Adult (n=6) 50.00 ±2.60 Total 171.74 ±7.03 n: number of individuals in each developmental stage Table 2. Feeding behavior in adult triatomines Time of feeding (min) Blood ingestion (mg) Blood ingestion rate (mg/min) M. pallidipennis (M) 26.24±11.10 445.60±265.00 * 18.66±9.71 M. pallidipennis (F) 34.09±14.05 760.40±317.00 * 23.28±6.05 M: adult males. F: adult females. Min: Minute. mg: milligram. mg/min: milligram per minute. Number of individuals/gender = 10. To compare time of feeding, blood ingestion and blood ingestion rate a non-para- metric Mann-Whitney test was used. To address the differences between the percentage of males and fe- males producing feces a Fisher exact test was used. Data are depicted as mean±standard deviations. An asterisk (*) indicates a significant result (P< 0.05) Table 3. Production of feces Time of defecation (min) % insects producing feces Frequency of defecation/meal M. pallidipennis (M) 29.69±7.98 70 0.80±0.63 M. pallidipennis (F) 25.43±7.83 100 1.30±0.48 M: adult males. F: adult females. Min: minute. Number of individuals/gender = 10. Data are depicted as mean±standard deviations Fig. 1. Oviposition of Meccus pallidipennis during fasting period After a blood meal, eight couples were separated into individual plastic flasks for 3 weeks as previously described. (A) Total number of eggs; (B) Eggs per couple/week and (C) Average eggs per couple per day. To compare the differences in oviposition among weeks a Friedman test was used. The results were ex- pressed as means±standard error of the means. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 262–268 E Franzim-Junior et al.: Biology of … 266 http://jad.tums.ac.ir Published Online: September 30, 2018 Discussion Meccus pallidipennis was able to acclima- tize to environmental conditions other than those naturally found in Mexico, reinforcing its vector potential in a distant geographical area. In this study, the life cycle of M. palli- dipennis was similar to that observed in pre- vious studies with the same species (10, 11), despite the differences in food sources and en- vironmental conditions observed in the differ- ent studies. Notably, this insect has the shortest biological cycle among triatomines of the same genre (12, 13); however, even under the same laboratory conditions, life cycles can be dis- tinct in different populations from the same species (14). The heterogeneity of the popu- lations of M. pallidipennis was further demon- strate in a study that showed the impact of dif- ferent environments on the reproduction, feed- ing behavior and development of this species in distinct areas of Mexico (8). Short biolog- ical cycles are epidemiologically critical be- cause they represent the ability of a species to repopulate and reestablish colonies right after disturbances, such as those produced by insec- ticides. This scenario makes triatomines more competent and capable of interacting with a host, consequently increasing the risk of trans- mission of T. cruzi. When compared to the main species of vectors in Latin America, the biological cycle of M. pallidipennis was longer than that described for T. infestans and Rhod- nius prolixus, both reaching adult stage with- in less than 100 days (15, 16). However, some other species may have a longer life cycle, such as that observed in T. boliviana (252 days), from Bolivia (17), and T. carcavalloi (193 days), from the Southern region of Brazil (18). On the other hand, it was similar to the development of Panstrongylus megistus (19) and Eratyrus mucronatus (20). Feeding behavior is one of the key ele- ments that dictate the vector efficiency and biology of triatomines. During a blood meal, not only they are able to transmit T. cruzi but also to fulfill their physiological needs for sur- vival. Females are more sensitive to fasting pe- riods than males from the same species (21). Different factors could explain this character- istic, including the energy expended in ovipo- sition and the maintenance of larger bodies in adult females compared to their male counter- parts. Accordingly, we demonstrated that adult females ingested greater amounts of blood than males, which could be related to the higher physiological and energetic demand associated with females. However, no differences between genders were observed for the feeding dura- tion and rate of blood ingestion. Though the feeding duration for females was similar to that observed in males, the higher amount of blood ingested by females may suggest a great- er voracity to their hosts. The observation that females ingest higher amounts of blood than males could explain why they are also consid- ered better vectors, besides as a mechanism for greater feces production (9, 14). Unfortunately, we did not find any differences between the amount of blood ingested and the production of feces when the genders were compared. Ad- ditionally, because a positive relationship was already established between the influence of the fasting period and the production of feces (22), we believe the fasting period in our study (30 days) might have increased the time needed for defecation. Furthermore, as females are more sensitive to fasting, they also tend to be more sensitive to weight loss when compared to their male counterparts from the same species. This characteristic makes females less-efficient vec- tors when exposed to greater fasting periods. Despite the impact on production of feces, the fasting period did not seem to influence the pro- portion of insects defecating before or after feeding. In the absence of regular feeding pe- riods, either oviposition or fertility could be negatively influenced (23). However, in our study, fasting did not in- fluence oviposition. These results indicate that http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 262–268 E Franzim-Junior et al.: Biology of … 267 http://jad.tums.ac.ir Published Online: September 30, 2018 the period of and sensitivity to fasting may fluc- tuate according to the feeding behavior, and longer feeding periods seem to enhance the period of oviposition. Conclusion Feeding behavior has a great impact on the life cycle of triatomines, which may also in- fluence T. cruzi transmission. Furthermore, the higher amount of blood ingested by females than males reinforced their potential as vec- tors. Finally, because M. pallidipennis was able to reach sexual maturity and reproduce even under different and fluctuating environmental conditions, it seems reasonable to assume its potential as a vector in environments other than those naturally find in Mexico. Acknowledgments We thank Dr José Alejandro Martínez Ib- arra from Universidad de Guadalajara, Mex- ico, by the donation of M. pallidipenis spec- imens used in this study. This work was sup- ported by the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Coor- denação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-150075/2016-2). References 1. WHO (2010) First WHO report on ne- glected tropical diseases: working to overcome the global impact of neglected tropical diseases. In: Organization WH, edi- tor.: WHO Press. 2. Tyler KM, Engman DM (2001) The life cycle of Trypanosoma cruzi revisited. Int J Parasitol. 31(5–6): 472–481. 3. Schofield CJ (1994) Triatominae-biology and control. Eurocommunica Publica- tions, London. 4. Piesman J, Sherlock IA (1983) Factors con- trolling the volume of feces produced by triatomine vectors of Chagas' disease. Ac- ta Trop. 40(4): 351–358. 5. Ibarra-Cerdena CN, Sanchez-Cordero V, Townsend Peterson A, Ramsey JM (2009) Ecology of North American Tri- atominae. Acta Trop. 110(2–3): 178–186. 6. Cruz-Reyes A, Pickering-Lopez JM (2006) Chagas disease in Mexico: an analysis of geographical distribution during the past 76 years-a review. Mem Inst Os- waldo Cruz. 101(4): 345–354. 7. Ramsey JM, Alvear AL, Ordonez R, Munoz G, Garcia A, Lopez R, Leyva R (2005) Risk factors associated with house in- festation by the Chagas disease vector Triatoma pallidipennis in Cuernavaca metropolitan area, Mexico. Med Vet En- tomol. 19(2): 219–228. 8. Martinez-Ibarra JA, Nogueda-Torres B, Salazar-Schettino PM, Vences-Blanco MO, de la Torre-Alvarez FJ, Montanez- Valdez OD (2014) Differences on bio- logical attributes of three populations of Meccus pallidipennis Stal (Hemiptera: Reduviidae). J Vector Borne Dis. 51(1): 22–26. 9. Martínez-Ibarra JA, Novelo-López M (2004) Blood meals to molt, feeding time and postfeeding defecation delay of Meccus pallidipennis (STÅL, 1872) (Hemiptera: Reduviidae) under laboratory conditions. Folia Entomol. 43(3): 313–319. 10. Martinez-Ibarra JA, Katthain-Duchateau G (1999) Biology of Triatoma palli- dipennis Stal 1945 (Hemiptera: Reduvi- idae: Triatominae) under laboratory con- ditions. Mem Inst Oswaldo Cruz. 94(6): 837–839. 11. Martinez-Ibarra JA, Nogueda-Torres B, Salazar-Montano LF, Garcia-Lino JC, Arroyo-Reyes D, Hernandez-Navarro JA (2017) Comparison of biological fitness in crosses between subspecies of Mec- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2018, 12(3): 262–268 E Franzim-Junior et al.: Biology of … 268 http://jad.tums.ac.ir Published Online: September 30, 2018 cus phyllosomus (Hemiptera: Reduvi- idae: Triatominae) in southern Mexico. Insect Sci. 24(1): 114–121. 12. Martinez-Ibarra JA, Grant-Guillen Y, Mar- tinez-Grant DM (2003) Feeding, defe- cation, and development times of Mec- cus longipennis Usinger, 1939 (Hemip- tera: Reduviidae: Triatominae) under la- boratory conditions. Mem Inst Oswaldo Cruz. 98(7): 899–903. 13. Martinez-Ibarra JA, Alejandre-Aguilar R, Torres-Morales A, Trujillo-Garcia JC, Nogueda-Torres B, Trujillo-Contreras F (2006) Biology of three species of the Meccus phyllosomus complex (Hemip- tera: Reduviidae: Triatominae) fed on blood of hens and rabbits. Mem Inst Oswaldo Cruz. 101(7): 787–794. 14. Martinez-Ibarra JA, Nogueda-Torres B, Garcia-Benavidez G, Vargas-Llamas V, Bustos-Saldana R, Montanez-Valdez OD (2012) Bionomics of populations of Mec- cus pallidipennis (Stal), 1872 (Hemip- tera: Reduviidae) from Mexico. J Vec- tor Ecol. 37(2): 474–477. 15. Carcavallo RU, Martínez A (1972) Life cycles of some species of Triatoma (He- miptera: Reduvidae). Can Entomol. 104 (05): 699–704. 16. Luz C, Fargues J, Grunewald J (1999) Development of Rhodnius prolixus (He- miptera: Reduviidae) under constant and cyclic conditions of temperature and humidity. Mem Inst Oswaldo Cruz. 94 (3): 403–409. 17. Duran P, Sinani E, Depickere S (2014) Biological cycle and preliminary data on vectorial competence of Triatoma boliviana in laboratory conditions. Ac- ta Trop. 140: 124–129. 18. Cardozo-de-Almeida M, Neves SC, Al- meida CE, Lima NR, Oliveira ML, San- tos-Mallet JR, Goncalves TC (2014) Bi- ology of Triatoma carcavalloi Jurberg, Rocha and Lent, 1998 under laboratory conditions. Rev Soc Bras Med Trop. 47 (3): 307–312. 19. Barbosa SE, Soares RP, Pires HH, Dio- taiuti L (2001) Experimental evidence for a demographic cline in Panstrongylus megistus populations. Mem Inst Os- waldo Cruz. 96(6): 773–775. 20. Monte GL, Tadei WP, Farias TM (2014) Ecoepidemiology and biology of Era- tyrus mucronatus Stal, 1859 (Hemip- tera: Reduviidae: Triatominae), a syl- vatic vector of Chagas disease in the Brazilian Amazon. Rev Soc Bras Med Trop. 47 (6): 723–727. 21. Costa MJ, Perondini AL (1973) Resist- ence of Triatoma brasiliensis to fast- ing. Rev Saude Publica. 7(3): 207–217. 22. Trumper EV, Gorla DE (1991) Density- dependent timing of defaecation by Triatoma infestans. Trans R Soc Trop Med Hyg. 85(6): 800–802. 23. Braga MV, Lima MM (2001) Effects of food deprivation levels on the oogene- sis of Panstrongylus megistus. Rev Saude Publica. 35(3): 312–314. http://jad.tums.ac.ir/ https://www.ncbi.nlm.nih.gov/pubmed/?term=Revista+de+Sa%C3%BAde+P%C3%BAblica.+35%3A+312%E2%80%93314. https://www.ncbi.nlm.nih.gov/pubmed/?term=Revista+de+Sa%C3%BAde+P%C3%BAblica.+35%3A+312%E2%80%93314.