Parasitol Res (2014) 113:4141–4149 DOI 10.1007/s00436-014-4086-3

ORIGINAL PAPER

Exsheathment and midgut invasion of nocturnally subperiodic Brugia malayi microfilariae in a refractory vector, Aedes aegypti (Thailand strain) N. Intakhan & N. Jariyapan & S. Sor-suwan & B. Phattanawiboon & K. Taai & W. Chanmol & A. Saeung & W. Choochote & P.A. Bates

Received: 1 May 2014 / Accepted: 14 August 2014 / Published online: 21 August 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Exsheathment and midgut invasion of nocturnally subperiodic Brugia malayi microfilariae were analyzed using light and scanning electron microscopy in a refractory vector, Aedes aegypti (Thailand strain). Results showed that exsheathed microfilariae represented only approximately 1 % of the total microfilaria midguts dissected at 5-min postinfected blood meal (PIBM). The percentage of exsheathed microfilariae found in midguts progressively increased to about 20, 60, 80, 90, and 100 % at 1-, 2–5-, 6–12-, 18–36-, and 48-h PIBM, respectively. Importantly, all the microfilariae penetrating the mosquito midguts were exsheathed. Midgut invasion by the exsheathed microfilariae was observed between 2- and 48-h PIBM. SEM analysis revealed sheathed microfilariae surrounded by small particles and maceration of the microfilarial sheath in the midguts, suggesting that the midguts of the refractory mosquitoes might have protein(s) and/or enzyme(s) and/or factor(s) that induce and/or accelerate exsheathment. The microfilariae penetrated the internal face of the peritrophic matrix (PM) by their anterior part and then the midgut epithelium, before entering the hemocoel suggesting that PM was not a barrier against the microfilariae migrating towards the midgut. Melanized microfilariae were discovered in the hemocoel examined at 96-h PIBM suggesting that This publication is dedicated to the memory of our esteemed colleague, Professor Wej Choochote N. Intakhan : N. Jariyapan (*) : S. Sor-suwan : B. Phattanawiboon : K. Taai : W. Chanmol : A. Saeung : W. Choochote Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand e-mail: [email protected] P. Bates Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster LA1 4YQ, United Kingdom

the refractory mosquitoes used melanization reactions against this parasite. This study provided evidence that A. aegypti (Thailand strain) has refractory mechanisms against B. malayi in both midgut and hemocoel. Keywords Aedes aegypti . Brugia malayi . Refractory . Midgut . Exsheathment

Introduction Lymphatic filariasis is a tropical disease caused by one of three parasitic nematodes, Wuchereria bancrofti, Brugia malayi, or Brugia timori. These filarial parasites are transmitted by several mosquitoes within the genera Mansonia, Anopheles, Culex, and Aedes (Schacher 1962; Guptavanij et al. 1978; Trpis 1981; Chang et al. 1991; Bangs et al. 1995; Kumar et al. 1998; Lek-Uthai and Tomoen 2005; Wada 2011). In competent vectors, the parasites continue their life cycle when microfilariae are taken up by female mosquitoes during blood feeding on an infected host. For normal development to proceed in such competent vectors, microfilariae must penetrate the mosquito midgut and traverse the hemocoel to invade the thoracic muscle cells, and then develop to the infective third larval stage. These migrate to the head and proboscis and enter a new host by penetrating the labellum of the proboscis when the mosquito takes another blood meal. Various factors have been reported that may inhibit or prevent filarial infection and successful development in the vector, and singly or in combination, these have been proposed to contribute to refractoriness. These factors include both physical obstacles (cibarial and pharyngeal armatures in the foregut) and physiological characteristics (peritrophic matrix formation and blood clotting in the midgut, direct toxicity,

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and melanization/encapsulation responses in thoracic muscles) of the vector, and are manifested as compatibility/ incompatibility of geographical strain variations in parasite and vector, and influenced by natural selection and adaptation in both parasite strain and vector species (Nelson 1964, Ewert 1965; Denham and McGreevy 1977; Owen 1979; Oda and Wada 1980; Townson and Chaithong 1991; Nayar and Knight 1995; Bangs et al. 1995; Christensen et al. 2005; Magalhaes et al. 2008). Blood-dwelling lymphatic microfilariae are usually sheathed, being enclosed in the remnant of the egg membrane, but this sheath is shed during development in the vector. In the midgut, the first vector microenvironment that filarial parasites encounter, early studies suggested that some microfilariae exsheathed either in the midgut itself (Esslinger 1962; Ewert 1965; Denham and McGreevy 1977) or during penetration of the midgut wall (Yamamoto et al. 1983; Christensen and Sutherland 1984; Agudelo-Silva and Spielman 1985; Perrone and Spielman 1986). Exsheathment preceding migration from the midgut has been considered by some authors as a prerequisite for the complete development of certain microfilariae in their insect vectors (Esslinger 1962; Ewert 1965; Santos et al. 2006). However, most previous studies of the development of Brugia parasites in competent vectors have only been performed using a susceptible strain of Aedes aegypti mosquitoes (black-eyed Liverpool strain) (Erickson et al. 2009), and often, these reached different conclusions. For example, Agudelo-Silva and Spielman (1985) studied the migration of B. malayi microfilariae in the black-eyed Liverpool strain, and concluded that the microfilariae penetrated the midgut wall of the mosquito vector while still sheathed, and that the sheath remained protruding from the gut when entering the hemocoel, thus arguing against exsheathment as a prerequisite for migration. On the other hand, Chen and Shin (1988) have suggested that the exsheathment of Brugia pahangi microfilariae occurs both in the midgut and hemocoel of susceptible (Liverpool) and refractory (Bora-Bora) strains of Ae. aegypti. The peritrophic matrix (PM) is secreted by the midgut epithelium upon blood feeding and encloses the blood meal, and this has to be transversed by microfilariae before they can access the midgut wall. It has been suggested that the PM is a barrier preventing microfilariae migrating towards the midgut and contributing to refractoriness (O’Connor and Beatty 1936; Iyengar 1936). In Culex pipiens pipiens, ingested B. malayi and B. pahangi microfilariae perish in the midgut soon after feeding, suggesting that the inability of C. p. pipiens to support the development and transmission of Brugia spp. occurs at the level of the midgut (Ewert 1965; Michalski et al. 2010). Christensen and Sutherland (1984) have reported that B. pahangi microfilariae freely traverse the PM of susceptible Ae. aegypti mosquitoes, and in addition, approximately 75 % retained their sheaths after midgut penetration and

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exsheathment of the microfilariae rarely occurred within the midgut. Further, Jariyapan et al. (2013) have recently demonstrated that in a susceptible vector, Ochlerotatus togoi (formerly known as Aedes togoi), only sheathed microfilariae of nocturnally subperiodic B. malayi invade across the PM through the midgut wall and these reach the hemocoel with their sheaths intact. Various previous studies examining filarial parasites in refractory (Malaya, Bora-Bora, and Bangkok) Ae. aegypti strains have been performed (Kershaw et al. 1961; Chaithong 1976; Chen and Shin 1988; Pothikasikorn et al. 2008). However, very little is known about selective barriers for Brugia development at the midgut level in refractory Ae. aegypti. Therefore, the current study was undertaken in which a systematic investigation of the exsheathment and invasion of nocturnally subperiodic B. malayi microfilariae within the midgut of a refractory vector, Ae. aegypti (Thailand strain), was performed.

Materials and methods Mosquitoes Ae. aegypti (Thailand strain) was used in this study. The mosquitoes were maintained at standard lighting conditions of 12-h light/dark, relative humidity of 70–80 % and temperature of 27 °C (±2 °C). Adult mosquitoes were provided with a 10 % (w/v) sucrose solution until they were 5–7 days old. Then, they were fasted for 12 h prior to feeding on anesthetized mice. After that, the mosquitoes were allowed to mate. The gravid female mosquitoes were encouraged to oviposit in a plastic cup of natural water with wet filter paper lining the inside. After hatching, 100 first instar larvae were placed in a white plastic tray (25×35×6 cm) containing 1,500 ml of natural water. The larvae were fed on fish food once a day. The resulting pupae were transferred to a plastic cup containing natural water and placed in a cage for emergence. These mosquitoes have been colonized continuously under laboratory conditions for several generations in the insectary of the Department of Parasitology, Faculty of Medicine, Chiang Mai University, since 2012. This mosquito strain was found to be refractory to nocturnally subperiodic B. malayi when previously tested. In the present study, this phenotype was confirmed before performing experiments. A group of female mosquitoes was infected as described below and dissected 14 days post-infected blood meal (PIBM) after feeding on B. malayi-infected blood and none were found infected Source of Brugia malayi microfilariae The nocturnally subperiodic B. malayi parasite strain originated from a 20-year-old female resident of Bang Paw district,

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Narathiwat province, southern Thailand. From 1982 until 1986, the strain was maintained by experimental infection of domestic cats at the Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. Then, the strain was transferred to Mongolian jirds (Meriones unguiculatus), and has since been maintained at the animal house of the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand (Choochote et al. 1986; Saeung and Choochote 2013).

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and fluids were thoroughly teased apart in PBS, allowed to air dry, fixed with absolute methanol, and Giemsa-stained. The microfilariae in each sample were counted and examined for the presence or absence of their sheaths under a light microscope. Photographs of the microfilariae were taken using a digital camera (Cannon, Tokyo, Japan) attached to the light microscope. Triplicate experiments were performed and counts were rechecked by more than one observer. Preparation of samples for scanning electron microscopy

Preparation of blood containing Brugia malayi microfilariae and infection of mosquitoes The jirds were anesthetized deeply with ethylene ether and inoculated intraperitoneally with infective larvae of B. malayi. The microfilariae were collected after at least 3 months (Choochote et al. 1991) by injecting 3 ml of Hank’s balanced salt solution (HBSS, pH 7.2–7.4) into the peritoneal cavity before withdrawing by peritoneal washing. A 0.5-ml volume of peritoneal washings enriched with microfilariae was mixed with 10 ml of human-heparinized blood (10 units of heparin/ ml of blood), which had been taken from donors. The microfilarial density was then adjusted to approximately 250 microfilariae/20 μl in human-heparinized blood, which was used for artificial mosquito feeding. The reason for this adjustment was based on previous experiments that yielded satisfactory susceptibility of O. togoi to nocturnally subperiodic B. malayi (susceptibility rates of 70–95 %, Jariyapan et al. 2013). This agreed with experiments reporting the susceptibility of Anopheles sinensis to periodic B. malayi using a microfilarial density of 5, 10, 20, and 50 microfilariae/ 20 μl, with susceptibility rates of 30, 65, 93, and 100 %, respectively (Luo and Qu 1990). Three-day-old adult females of Ae. aegypti (fasted for 12 h) were infected by artificial feeding on blood containing B. malayi microfilariae, using the techniques and apparatus described by Chomcharn et al. (1980).

Engorged mosquitoes were dissected as described above and 10 samples from each time point were processed for scanning electron microscopy (SEM). Dissected midguts were fixed overnight with a solution of 2.5 % glutaraldehyde in PBS, pH 7.4 at 4 °C to accomplish primary fixation. The samples were then rinsed twice with PBS at 10-min intervals and postfixed for 2 h in a solution of 1 % osmium tetroxide. Postfixation was followed by rinsing twice with PBS. Water was replaced in the specimens with alcohol by subjecting them to alcoholic concentrations of 30, 50, 70, 80, 90, and 95 %. Then, the specimens were placed in absolute alcohol for two 12-h periods. After that, specimens were placed in acetone for 2 h, subjected to critical point drying, and attached to aluminum stubs with double-sided tape. Finally, they were coated with gold in a sputter-coating apparatus before being viewed with a scanning electron microscope (JEOL JSM-5910LV, JEOL Ltd., Japan). To observe the interface between the midgut surface and the microfilarial infected blood meal, some fixed samples were fractured before being coated with gold, while others were gently opened and their contents were washed out before fixation with PBS. Ethical clearance The protocols described above were approved by the Animal Ethics Committee of the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.

Exsheathment studies and preparation of samples for light microscopy To determine if exsheathment of B. malayi microfilariae occurred within the midgut, engorged mosquitoes were dissected and their midguts recovered in phosphate-buffered saline solution (PBS) at different time points after the blood meal (5 min and 1, 2, 3, 4, 5, 6, 12, 18, 24, 36, 48, 72, and 96 h), taking care not to damage the midgut. Ten excised intact midguts were processed for each time point. Each individual midgut was carefully transferred to a new glass slide and then opened. The ingested blood meal from the midgut was used to prepare a thick blood film, which was dried out, dehemoglobinized, fixed with absolute methanol, and stained with Giemsa stain (pH 7.2). The remaining mosquito body

Results Analysis of exsheathment and midgut invasion of Brugia malayi microfilariae in Aedes aegypti using light microscopy Mosquitoes were infected by blood feeding and at various time points PIBM, were dissected; microfilariae with intact morphology were counted; and the presence/absence of the sheath were recorded (Fig. 1). Table 1 shows the average number and percentage of nocturnally sub-periodic B. malayi microfilariae in the midgut and hemolymph of Ae. aegypti at different time points PIBM ranging between 5 min and 96 h.

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Fig. 1 Representative images of B. malayi microfilariae with intact morphology in Ae. aegypti. a A sheathed microfilaria in the midgut.

Arrows indicate the microfilarial sheath. b An exsheathed microfilaria in the midgut. c An exsheathed microfilaria in the hemolymph

The average total number of microfilariae found in mosquitoes varied from approximately 34 to 48 per mosquito up until 36-h PIBM, but with no obvious trend in numbers over this time period. The highest number of microfilariae recorded in a single mosquito up to 36-h PIBM was 117, and the lowest was 12. However, from 48-h PIBM onwards, there was a clear decrease in numbers, the average number of microfilariae found decreased more than twofold to 48 h, and further decreased at 72- and 96-h PIBM. In addition to changes in the total number of microfilariae per mosquito PIBM, their distribution also changed as some microfilariae crossed the midgut wall and were found in the hemolymph. The earliest time point microfilariae were found in hemolymph was after only 2 h, the highest numbers were recorded at 24–48 h, but at all points PIBM, these only represented small numbers of microfilariae. All the microfilariae used in experiments were sheathed at the beginning, and accordingly at 5 min PIBM (the first sampling point) almost all had still retained their sheath,

and exsheathed microfilariae in the midguts were less than 1 % of the total at this time point. However, at later time points PIBM the average percentage of exsheathed microfilariae found in midguts progressively increased to about 20, 60, 80, 90, and 100 % at 1-, 2–5-, 6–12-, 18–36-, and 48-h PIBM, respectively. Microfilariae were observed in the mosquito hemolymph between 2- and 96-h PIBM, and importantly all of these microfilariae found in the hemolymph were exsheathed. These results indicate that the microfilariae are unable to retain their sheath until they reach the hemocoel in this refractory mosquito, and appear to lose their sheaths in the midgut prior to or in the act of crossing the midgut wall. Analysis of exsheathment and midgut invasion of Brugia malayi microfilariae in Aedes aegypti using SEM To investigate the exsheathment of the microfilariae in the midguts and their penetration across the Ae. aegypti in greater

Table 1 Average number and percentage of microfilariae (mff) of NSP B. malayi in the midgut and hemolymph in Ae. aegypti at different times PIBM based on 30 infected mosquitoes per time point from triplicate experiments (n=10/mosquito/time point/experiment; 450 total) Time PIBM

5 1 2 3 4 5 6

min h h h h h h

12 18 24 30 36 48 72 96

h h h h h h h h

Average no. of total mff found per mosquito (range)

Average no. of mff found in midgut per mosquito (range)

Average no. of mff found in hemolymph per mosquito (range)

Mff found in midgut per mosquito

Mff found in hemolymph per mosquito

Average no. of sheathed mff (%)

Average no. of Average no. exsheathed of sheathed mff (%) mff (%)

Average no. of exsheathed mff (%)

34.33 (12–60) 38.87 (20–66) 46.40 (25–76) 42.33 (24–67) 43.93 (18–67) 38.93 (27–55) 42.40 (19–83)

34.33 (12–60) 38.87 (20–66) 45.53 (24–76) 40.73 (24–65) 41.20 (18–63) 35.80 (25–52) 39.47 (19–77)

0 0 0.87 (0–2) 1.60 (0–4) 2.73 (0–5) 3.13 (1–7) 2.93 (0–6)

34 (99.04) 30.13 (77.51) 18.07 (39.69) 15.53 (38.13) 16.07 (39.00) 14.33 (40.03) 9.13 (23.13)

0.33 (0.96) 8.73 (22.49) 27.47 (60.31) 25.20 (61.87) 25.13 (61.00) 21.47 (59.97) 30.33 (76.87)

0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

0 (0) 0 (0) 0.87 (100) 1.60 (100) 2.73 (100) 3.13 (100) 2.93 (100)

42.73 (21–71) 46.00 (22–63) 48.13 (26–117) 36.27 (18–61) 34.20 (15–49) 17.27 (10–28) 5.53 (2–10) 2.60 (1–6)

38.13 (18–67) 39.00 (11–60) 36.87 (20–102) 28.87 (10–57) 23.20 (9–36) 11.13 (7–17) 0 0

4.60 (2–9) 7.00 (3–11) 11.27 (4–22) 7.40 (3–11) 11.00 (6–20) 6.13 (1–11) 5.53 (2–10) 2.60 (1–6)

8.00 (20.98) 4.00 (10.27) 3.33 (9.03) 2.80 (9.70) 1.80 (7.76) 0 (0) 0 (0) 0 (0)

30.13 (79.02) 35.00 (89.73) 33.53 (90.97) 26.07 (90.30) 21.40 (92.24) 11.13 (100) 0 (0) 0 (0)

0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

4.60 (100) 7.00 (100) 11.27 (100) 7.40 (100) 11.00 (100) 6.13 (100) 5.53 (100) 2.60 (100)

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detail, SEM was used. SEM analysis at early time points revealed that some sheathed microfilariae were surrounded by small granular particles, suggestive of some kind of degradative process (Fig. 2a). Breaking up or maceration of the microfilarial sheath was also observed in the midguts of the mosquitoes dissected at 3-h PIBM (Fig. 2b, d). Between 5min and 4-h PIBM, exsheathed microfilariae were also observed inside the midgut lumen of mosquitoes, either in contact with or close to the PM (Fig. 2e, f). Invasion of the B. malayi microfilariae into the hemocoel was observed in SEM samples taken 2–4-h PIBM. The microfilariae could be seen to have penetrated the internal face of the PM by their anterior part, and then the midgut epithelium before entering the hemocoel (Fig. 3). There was no evidence that the PM functioned as a barrier to the migration of the microfilariae out of the blood meal at any times PIBM that they were examined. All B. malayi microfilariae seen penetrating the midgut epithelium were seen to be exsheathed in SEM (Fig. 3), confirming the results from light microscopy (LM) observations. In contrast, Fig. 4a–f demonstrate the presence of sheathed microfilariae in the lumens of the midguts examined during 1–36-h PIBM. Melanization of some microfilariae were observed in the hemocoel at 96-h PIBM (Fig. 5). No

Fig. 3 SEM micrographs of fractured abdominal midguts showing the invasion process of microfilariae. Specimens are from the midgut lumen and hemocoel at 4-h PIBM. a, b Exsheathed microfilariae (arrows) close to the PM. c Exsheathed microfilariae (arrows) crossing the PM. d Fractured region showing exsheathed microfilariae (arrow) during invasion into the PM. e An exsheathed microfilaria (arrow) penetrating across the PM and epithelium into the hemocoel and lying on the external surface (Ex) of the midgut

mature L3 larval stages were observed in the thoracic muscles or elsewhere in mosquitoes examined at 96-h PIBM.

Discussion

Fig. 2 SEM micrographs of abdominal midguts showing the exsheathment of microfilariae. a A representative image of a sheathed microfilariae (arrow) surrounded by small particles in the midgut lumen observed in a midgut dissected at 1-h PIBM. b–d Representative examples of maceration of the sheaths (arrows) of microfilariae in midguts dissected at 3-h PIBM. e–f Representative examples of exsheathed microfilariae (arrows) inside the blood meal (BM) in the midgut dissected at 3-h PIBM

Exsheathment is an important and necessary process for the development of microfilariae into L3 infective larvae (Ewert 1965). In the present study, exsheathment of B. malayi microfilariae was found to occur in the midgut of the refractory Ae. aegypti (Thailand strain) from 5-min to 48-h PIBM and before penetration into the hemocoel. Although we cannot completely exclude the possibility of exsheathment occurring during penetration of the midgut wall in some microfilariae, the finding of large numbers of exsheathed microfilariae in the

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Fig. 4 SEM micrographs showing sheathed microfilariae in the midgut lumen. a–f Representative examples from midguts dissected at 1-, 3-, 12-, 24-, and 36-h PIBM, respectively

midgut and SEM observations of only exsheathed microfilariae penetrating the gut wall, both indicate that most if not all exsheathment occurred in the midgut. These results, using a refractory strain of Ae. aegypti contrast with those for B. malayi microfilariae in the susceptible Black eye Liverpool strain, which do not exsheath until they penetrate the midgut wall (Yamamoto et al. 1983; Agudelo-Silva and Spielman

Fig. 5 An example of a melanized microfilaria recovered from the hemocoel. LM showing melanization occurred on a part of the microfilaria body (arrow)

1985; Perrone and Spielman 1986), and similarly also contrast with those in the susceptible vector O. togoi (Jariyapan et al. 2013). Although Chen and Shin (1988) have reported that B. pahangi microfilariae tend to carry their sheaths into the hemocoel of both susceptible (Liverpool) and refractory (Bora-Bora) strains of Ae. aegypti within 2-h after feeding on a filarial-infected rat, some of the microfilariae were exsheathed in the midgut of both strains. Perrone and Spielman (1986) have demonstrated that exsheathment of B. malayi microfilariae in the Ae. aegypti (Black eye Liverpool strain) is enhanced by movement against the constricting midgut wall. The sheath of B. pahangi is suggested to be weakened or broken at the anterior end of the microfilariae during penetration of the midgut of Ae. aegypti, Black eye Liverpool strain (Christensen and Sutherland 1984). In the current study, sheathed B. malayi microfilariae in the midgut were surrounded by small particles and maceration of the sheath was also observed in the Ae. aegypti midguts, suggesting that the midguts of the refractory mosquitoes might have protein(s) and/or enzyme(s) and/or other factor(s) that induce and/or accelerate degradation of the sheath and consequent exsheathment. To our knowledge, this is the first report of such small particles and maceration of the microfilarial sheath

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in the midgut of a refractory strain of Ae. aegypti. It will be important to investigate other refractory strains of Ae. aegypti and/or mosquito species to determine whether similar observations are made as in the Thailand strain and determine whether this is a general mechanism. During blood meal digestion, several proteolytic enzymes are released in the Ae. aegypti midgut to degrade blood meal proteins into peptides and amino acids, for example, trypsins (Felix et al. 1991; Barillas-Mury and Wells 1993; Kalhok et al. 1993), chymotrypsins (Jiang et al. 1997; Bian et al. 2008), aminopeptidases (Billingsley and Rudin 1992; Noriega et al. 2002), carboxypeptidases (Edwards et al. 2000; Isoe et al. 2009a), and serine proteases (Isoe et al. 2009b). TchankouoNguetcheu et al. (2010) have analyzed the response of Ae. aegypti midguts to infection with Chikungunya and dengue 2 viruses. This study reported that differentially regulated proteins in response to viral infection include structural, redox, regulatory proteins, and enzymes for several metabolic pathways. Some of these proteins, such as those with antioxidant properties, are probably involved in cell protection. Tchankouo-Nguetcheu et al. (2010) have also proposed that the modulation of other proteins such as transferrin, hsp60, and alpha glucosidase may favor virus survival, replication, and transmission. Recently, the first proteomic analysis of the Aedes albopictus midgut has been reported (Saboia-Vahia et al. 2012). Fifty-six proteins were identified with sequence similarity to entries from the Ae. aegypti genome. Five functional networks among the identified proteins include a network for carbohydrate and amino acid metabolism, a group associated with ATP production, and a network of proteins that interact during detoxification of toxic free radicals, among others. Several enzymes, for example, endopeptidase and papaya extract protease, as well as calcium ions, pH conditions, temperature, and ivermectin have been proved to be effective in stimulating the exsheathment of microfilariae of B. pahangi, B. malayi, Litomosoides carinii, and W. bancrofti (Weinstein 1963; Devaney and Howells 1979; Rao et al. 1992). However, to date, no specific protein(s) and/or enzyme(s) and/or factor(s) involved in exsheathment of microfilariae in the midgut of Ae. aegypti have been identified. Therefore, further work to identify and characterize the small particles and maceration of the B. malayi microfilarial sheath in the midgut of the refractory Ae. aegypti (Thailand strain) is required. Further, as exsheathment and midgut penetration are important processes for development of microfilariae, the potential interruption of transmission by interfering with the parasite molecules involved in exsheathment and penetration could be important in the development of transmission control strategies. Exsheathed B. malayi microfilariae were observed penetrating the PM and midgut epithelium into hemocoel of the Ae. aegypti (Thailand strain) suggesting that the PM of the refactory strain was not a barrier against migration of the

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microfilariae. These observations are in accordance with several previous studies that have reported the PM is not a barrier against migration of Brugia microfilariae to the hemocoel in susceptible Ae. aegypti (Black eye Liverpool strain) and O. togoi (Ewert 1965; Christensen and Sutherland 1984; Jariyapan et al. 2013). Melanization is an innate immune response mounted against foreign organisms in insects (Christensen et al. 2005, Zou et al. 2010; Thomas et al. 2011; Sakamoto et al. 2011; Tokura et al. 2014). In this study, melanized B. malayi microfilariae were discovered in the hemocoel at 96-h PIBM suggesting that the Ae. aegypti (Thailand strain) used melanization reactions against this parasite. An explanation might be that as the microfilariae were too large to be phagocytosed by hemocytes, therefore, the mosquitoes synthesized melanin as a defensive reaction against molecules on the surface of the body of the exsheathed microfilariae. Consequently, the microfilariae were unable to develop further to a larval stage. Melanization of sheaths and microfilariae of B. malayi have also been observed in Anopheles quadrimaculatus and Ae. aegypti (Nayar and Knight 1995). Recently, Saeung et al. (2013) have reported melanization of B. malayi L1 larvae in refractory vectors, Anopheles paraliae, An. sinensis, and Anopheles nitidus. Two refractory mechanisms, direct toxicity and/or melanotic encapsulation, have been suggested to be involved in the refractoriness of development in the thoracic muscles of the mosquitoes (Saeung et al. 2013). In C. p. pipiens, Michalski et al. (2010) have demonstrated that B. pahangi and B. malayi microfilariae in the mosquito midgut have compromised motility, sharp bends in the body, and internal damage, revealed as the presence of many fluid or carbohydrate-filled vacuoles in the hypodermis, body wall, and nuclear column, indicating that the C. p. pipiens midgut has factor(s) that damage the microfilariae. In conclusion, the present study is the first report of small particles and maceration on the microfilarial sheath in the midgut of the refractory strain of Ae. aegypti. Results demonstrated that the exsheathment of microfilariae of B. malayi occurred only in the midgut of the Ae. aegypti Thailand strain and the PM was not a barrier against the microfilariae migrating from the midgut to the hemocoel. In addition, melanized microfilariae were discovered in the hemocoel suggesting that the refractory mosquitoes used melanization reactions against this parasite. The overall results indicate that Ae. aegypti (Thailand strain) has refractory mechanisms against B. malayi that operate in both midgut and hemocoel. Acknowledgments This work was financially supported by the Thailand Research Fund (TRF Senior Research Scholar: RTA5480006 to WC, subproject to NJ), and the Faculty of Medicine Endowment Fund, Chiang Mai University.

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