REAÇÕES IMUNOLÓGICAS DE Aedes aegypti LINNAEUS, 1762, DE Aedes albopictus SKUSE, 1894 E Cx J Arthropod-Borne Dis, March 2018, 12(1): 94–99 G Aparecida de Carvalho et al.: Melanization of … 94 http://jad.tums.ac.ir Published Online: March 18, 2018 Short Communication Melanization of Dirofilaria immitis Larvae in Different Culicid Species *Gílcia Aparecida de Carvalho 1, Rafael Antonio Nascimento Ramos 2, Rafael Trindade Maia 3, Carlos Fernando Salgueirosa de Andrade 4, Leucio Câmara Alves 2 1Academic Unit of Garanhuns, Federal Rural University of Pernambuco, Garanhuns, Brazil 2Department of Veterinary Medicine, Federal Rural University of Pernambuco, Recife, Brazil 3Department of Biological Sciences, Federal University of Campina Grande, Sumé, Brazil 4Department of Animal Biology, Biology Institute, State University of Campinas, Campinas, Brazil (Received 13 Oct 2015; accepted 3 Feb 2018) Abstract Background: Dirofilaria immitis is an important filarioid transmitted by culicids. The vector role of these arthropods may be influenced by biological events as melanization against D. immitis larvae. This study aimed to detect the occurrence of melanization in Culex quinquefasciatus, Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus experimentally infected with D. immitis. Methods: Five populations (Cx. quinquefasciatus RECIFE (P1), Ae. albopictus RECIFE (P2), Ae. aegypti RECIFE (P3), Ae. aegypti CAMPINAS (P4) and Ae. aegypti ROCKEFELLER (P5)) were artificially fed with infected blood containing D. immitis microfilariae. Ten mosquitoes per day from each population were dissected for 14 days. Results: Melanized larvae of D. immitis were observed in all population except in P3. The period in which melanized larvae were found varied from the third to the 14th day post-infection. Difference in the number of these larvae was observed between P5 and P2–P3 (P< 0.01), and between P5 and P4 (P< 0.05). Third-stage larvae of D. immitis were detected in all population 14 d post-infection, except in P3, which presented high mortality 24 h post- infection. The melanization observed in the populations herein studied did not indicate refractory since third-stage larvae were observed at the end of the experiment. Most likely, this immune response aimed to control the number of larvae, in order to maintain the equilibrium between vector-parasite. Conclusion: The melanization did not seem to be a limiting factor to the development of this filarioid in these local Brazilian Culicidae populations. Keywords: Mosquitoes, Immune response, Dirofilariasis, Vector Introduction Dirofilaria immitis is an important nema- tode filarioid that causes a severe infection in dogs commonly known as heartworm dis- ease. Although, other animal species as wild carnivores, cats and humans may be affect- ed, dogs present a high epidemiological rele- vance (1). This filarioid is transmitted by cu- licid vectors, being species belonging to the genus Culex, Aedes and Anopheles the most important (2-5). The vector role of Culicidae species may be influenced by biological events as immune re- sponses against D. immitis microfilariae. Indeed, different types of immune response against parasites and pathogens affecting mosquitoes have been studied in some species (e.g., Aedes aegypti, Ochlerotatus trivittatus, Anopheles quadrimaculatus, and Armigeres subalbatus) (6-10). For example, the melanization is an important immune event that occurs inside the mosquito vector. This phenomenon is char- acterized by an initial reaction involving the lysis of hemocytes close the surface of the parasite prior to the deposition of pigments (8, 11). The melanization may impair the flow of nutrients that are essential to the micro- filariae survival and development. Moreover, it may difficult the release of toxic oxygen *Corresponding author: Prof Gílcia A. Carvalho, E-mail: gilcia.acarvalho@yahoo.com http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 94–99 G Aparecida de Carvalho et al.: Melanization of … 95 http://jad.tums.ac.ir Published Online: March 18, 2018 metabolites causing the death of the parasite (12). The melanization of D. immitis larvae was reported in A. subalbatus, Ae. aegypti and Cx. quinquefasciatus (11, 13, 14). This immune response may reduce the vector ability of some populations of mosquitoes in transmit par- asites, including D. immitis (15). On the other hand, species belonging to the genus Aedes use this phenomenon to restrict the larval de- velopment, enhancing its potential as vector (16, 17). A melanization is an event that may vary depending on the population studied, and in Brazil, this phenomenon has been poorly studied in the local Culicidae population. Therefore, the aim of this study was to assess the occurrence of melanization in dif- ferent populations of culicids (Cx. quinque- fasciatus, Ae. albopictus and Ae. aegypti) ex- perimentally infected with D. immitis micro- filariae to evaluate the potential role of these species as vectors. In addition, biological im- plications of these findings have been dis- cussed. Materials and Methods Dirofilaria immitis microfilariae Infected blood containing D. immitis mi- crofilariae was obtained from a positive dog diagnosed at microscopic and molecular anal- yses. The animal was a three-yr-old male that lived in the metropolitan region of Recife (7°45'0"S and 34°51'0"W), state of the Per- nambuco, Brazil. Mosquito populations and experimental in- fection Five populations belonging to three dif- ferent culicid species were used in this study: Cx. quinquefasciatus RECIFE (P1), Ae. al- bopictus RECIFE (P2), Ae. aegypti RECIFE (P3), Ae. aegypti CAMPINAS (P4) and Ae. aegypti ROCKEFELLER (P5). Overall, 6000 female mosquitoes (1200 per each population, test group =900 and control group = 300) aged from three to seven days were used (18). The artificial blood meal was performed as previously described (4, 5). Brief- ly, mosquitoes were fed for two hours with infected blood containing about 2000 mi- crofilariae/ml of D. immitis. After blood meal, mosquitoes were maintained under controlled conditions of temperature (28±2 °C) and rel- ative humidity (>70%). Mosquito dissection and microscopic ex- amination Ten mosquitoes per day from each pop- ulation test were dissected during 14 days. Daily, dead mosquitoes were discarded and at the end of the experiment the remaining ones were dissected. Briefly, specimens were fixed on slides containing a drop of 0.9% physiological saline solution, dissected with a sterile scalpel and immediately examined under a light microscope (Olympus BX41 TF) at different magnifications. All stages of D. immitis larvae were morphologically iden- tified (19) and the presence of melanized lar- vae was recorded. Data analysis The percentage of infected mosquitoes, as well as number of melanized larvae, was calculated. The statistical analysis was per- formed through the Partitioning Qui-square test using the software BioEstat 2.0 (20). Results The overall results with the number of infected females and melanized larvae are shown in Table 1. Melanized larvae were de- tected in all population except in P3 and the highest number was detected in P5 followed by P1, P4, and P2, respectively. In general, the period in which these melanized larvae were found varied from the third to the 14th day post-infection. The developing stage in http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 94–99 G Aparecida de Carvalho et al.: Melanization of … 96 http://jad.tums.ac.ir Published Online: March 18, 2018 which they were observed is reported in Table 2. Difference in the number of melanized larvae was observed between P5 and P2 (P< 0.01), and between P5 and P4 (P< 0.05). Interestingly, third-stage larvae of D. im- mitis were detected in all population 14d post-infection, except in P3, which presented a high mortality (70.7%) 24 h post-infection. The P2 presented the first L3 nine days post- infection, whereas for P4 and P1 third-stage larvae were observed ten and 12d post-in- fection, respectively. Finally, for P5 D. im- mitis L3 were retrieved only 14d post-infec- tion, but melanized first-stage larvae were detected until the end of the experiment. The infected mosquitoes showed total or partial destruction of the cells of Malpighian tubules (MT) in the parasitized areas. The membrane of the cells presented damaged, most likely due to the migration of the larvae from the tubules to the head and probos- cides. During this migration, the reaction of melanization was not observed. During the whole study, the mortality of mosquitoes of control groups was approxi- mately 3% for all groups herein analyzed. Table 1. Infected females with Dirofilaria immitis microfilariae, and presence of melanized larvae P1 P2 P3 P4 P5 Females with D. immitis microfilariae (%)* 13.47 (111/824) 15.63 (131/838) 17.55 (149/849) 11.57 (95/821) 16.97 (149/878) Females with D. immitis melanized larvae ** (%) 10.81 a (12/111) 0.76 b (01/131) 0 b (0/149) 5.26 a, c (05/95) 19.46 a, d (29/149) (*Number of females infected/Number of females dissected, **Number of females with melanized larvae/Number of females infected. Different letters indicate statistically significant difference) Table 2. Developing stage of the melanized larvae in different populations analyzed First-stage Salsichoid and second-stage % (n/N) Third-stage P1 - 100 (12/12) - P2 - 100 (1/1) - P4 80 (4/5) - 20 (1/5) P5 68.9 (20/29) 31.1 (9/29) - Discussion In this study, the melanization of D. immitis larvae in different culicid populations and spe- cies was studied. The melanization that oc- curs in D. immitis larvae in some mosquitoes suggests that these specimens may be resistant to the parasitism by this filarioid, depending on the percentage of melanized larvae (16). Indeed, in populations such as Ae. aegypti ROCKEFELLER this phenomenon is more evident (21, 22). In general, the development of D. immitis larvae in culicids occurs in 14d, from micro- filariae to infective-stage larva. However, de- pending on the mosquito population, several biological events (e.g., blood coagulation, mi- crofilaria capture and melanization) may oc- cur to control the number of larvae in de- velopment inside the Malpighian tubules (5- 16). These mechanisms are important to reg- ulate the number of infected mosquitos, which http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2018, 12(1): 94–99 G Aparecida de Carvalho et al.: Melanization of … 97 http://jad.tums.ac.ir Published Online: March 18, 2018 will influence in their survival and vectorial competence (23). In the present study, almost all populations (except P3) presented melanized larvae, but in all of them (except P3) third-stage larvae of D. immitis were detected at the end of the experiment. Most likely, in this population microfilariae were destroyed at the first 24 h post-infection, therefore, no melanized larvae and infective-stage larvae were observed. This is an important finding, especially because all populations and species herein studied are susceptible and allows the development of D. immitis microfilariae; accordingly, these populations are considered potential vectors of D. immitis. The susceptibility of Ae. aegypti species for D. immitis infection is a characteristic con- trolled by the f1 gene (24). Females that pre- sent the patterns FiFi or Fifi are refractory to the infection. Indeed, these genes may influ- ence the physiology of Malpighian tubules, and consequently the development of the par- asite. Most likely, this genetic factor has an important role in the phenomenon of melani- zation, affecting the vector competence of culicids (8, 25). In fact, genetic differences may be observed in individuals of the same species (eg: Ae. aegypti) and different pop- ulations (24). These differences may be the cause of the findings of melanization herein reported for the same culicid species (ie, P4 and P5). This variation within a mosquito species contributes to enhancing the vector role of some populations from different ge- ographical areas (26). The structural alterations herein observed in infected mosquitoes may play an important role in the development of D. immitis larvae. The mortality of infected mosquitoes proba- bly may occur due to two reasons: i) the invasion of the MT cells by the microfilariae and ii) the escape of the infective larvae from the MT and their movement to the head and mouthparts (27). Therefore, when the par- asite load in the MT is higher, an excessive mortality (almost 100%) may occur (28, 29). In this study, the parasitism by D. immitis microfilariae had a great impact on females of P3, influencing the survival of specimens which dead (almost 70.7%) 24h post-infection. Conclusion The melanization observed in the pop- ulations herein studied did not indicate re- fractory, since third-stage larvae were detect- ed at the end of the experiment. 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