Abstract: Leishmaniasis, mainly a zoonotic disease vectored by sand flies, emerges as major public health concern in Sri Lanka. The role of the old world sandfly genus Sergentomyia in the disease transmission is emphasized in recent findings. Seven species belong to the subgenus Sergentomyia of this genus have been reported globally. Only one (Sergentomyia (Sergentomyia) punjabiensis) has been recorded so far from Sri Lanka. Three species viz. Se. (Ser.) punjabiensis, Se. (Ser.) pondicherriensis and Se. (Ser.) dentata were identified in a collection from three Cutaneous Leishmaniasis (CL) endemic localities in Sri Lanka. This is the first record of the presence of Se. (Ser.) pondicherriensis and Se. (Ser) dentate from the country. The detail morphology of both sexes are described along with the comparison with the existing literatures from the oriental region.
INTRODUCTION
Phlebotomine sand flies (Diptera: Psychodidae: Phlebotaminae) are of human health concern. They are vectors of human diseases such as sandfly fever (Tavana, 2007) and leishmaniasis (Alexander and Maroli, 2003). The sandflies are classified, based on the geographical distribution, in to two major groups namely, old world and new world flies. Old world sandflies include three genera viz. Phlebotomus, Chinius and Sergentomyia while the New World includes the genera viz. Brumptomyia, Lutzomyia and Warileya (Lane, 1993).
Only three sandfly species were named in 1905 and since then 700 sandfly species have been identified (Ilango, 2011). Various studies have been carried out in Sri Lanka since early 1900s to record the presence of sandflies in the country (Lewis, 1978). However these studies were not conclusive and there is no catalogue of Sri Lankan sandlfies.
The members of the genus Sergentomyia Franca and Parrot mostly prefer to feed on reptiles (Lewis, 1978; Artemiev, 1978). However, various research findings suggest that they could transmit various pathogens including Leishmania (Geevarghese et al., 2004; Maroli et al.,1988; Mukherjee et al., 1997). Leishmaniasis is an emerging public health threat in Sri Lanka. The first autochthonous Cutaneous Leishmaniasis (CL) case was reported from the country in 1992 (Athukorale et al., 1992). Since then, more than 2000 CL cases have been reported (Siriwardana et al., 2010). The first case of Visceral Leishmaniasis (VL) was reported from North Central Province in 2006. Many CL patients have been identified in many localities in the dry zone of the country. Some of the localities that were identified, like Matara, Hambantotta, Polannaruwa, Vavuniya, Padaviya, Siripura and Anurathapura where large number of patients have been recorded for (Abeygunasekara et al., 2007). The disease transmission to new localities is attributed to insufficient diagnostic facilities as well as lack of awareness (Ozbel et al., 2011).
The vector for either CL or VL in Sri Lanka is yet to be identified. Sand fly taxonomy and proper identification of species is crucial. This plays a major role in vectors species identification and also in the formulation of proper control measures (Singh et al., 2007). Therefore, a comprehensive taxonomic work is required to facilitate the perfect identification of sand fly species and to formulate an optimum vector control programme in Sri Lanka.
In this view, a study was carried out to record the presence of Sergentomyia species in the country and the results are reported with the evidence for the presence of three species of the subgenus Sergentomyia of which two are recorded for the first time from the country.
MATERIALS AND METHODS
The present investigation was carried out in leishmaniasis endemic localities in the districts of Vavuniya, Trincomalee and Anurathapura which are located in the dry zone of the country (Fig. 1). Sandflies were collected in and around the houses (including that of the leishmaniasis patients) by a mouth aspirator form 1700 to 0500 h from May to August 2010. Collected samples were transferred in eppendorf vials (1.5 mL) containing 70% ethyl alcohol and shifted to Zoology Department, University of Jaffna, where morphometric and meristic analysis was done. Each fly was processed and dissected following the conventional techniques (Young and Duncan, 1994; Aslamkhan and Aslamkhan, 2000). Whole specimen was temporarily mounted in distilled water to study the morphometric and meristic characteristics.
The flies were observed under a stereo microscope (Kyowa Model SE-L, Japan) and a monocular light microscope (Kyowa, Japan) equipped with a moving venire scale and an ocular micrometer. Permanent mounting of the whole specimen was done for some flies in Canada balsam after the alcohol treatment (Ko et al., 2008).
Photographs of the morphometric structures were taken with the Nikon Coolpix Digital Camera fixed to the microscopes. Camera Lucida was used to draw images. The measurement of the structures such as wing, wing venation (Radial (R) 1, 2, R 3, R 2+3 and R1 Overlap), halter, head, eye, labium, maxillary palp, antennal flagellomere (AF)-i,-, S. chaetica on AFi-v, thorax, femur, tibial, basitarsal segments of each appendages, abdomen, coxa, style (clasper), paramere, aedegal sheath, genital pump and aedegal filament was done with the ocular micrometer. The number of teeth found in both ventral and lateral sides of the maxillae was counted under the light microscope equipped with a camera (Olympus Model BX 51, Japan).
Identification of specimens was done with the available taxonomic keys (Lewis, 1978; Lane, 1993; Artemiev, 1978; Kakarsulemankhel, 2009; Srinivasan and Jambulingam 2010). Prepared permanent slides were deposited at the Department of Zoology, University of Jaffna.
Morphometric characters of the identified flies were compared with the available literatures from the South East Asian region (Lewis, 1978; Kakarsulemankhel, 2004a, b, 2009, Pringle, 1953; Perfiliev, 1933, 1960, 1968; Sinton, 1933) (Table 1-6) for identifying any variation in described morphometric and any new taxa.
| Fig. 1: | The sampling localities (P- Padavi Siripura in Trincomalee district; Pa- Padaviya in Anurathapura district; V- Vavuniya) |
RESULTS
Nineteen flies belonging to the genus Sergentomyia and subgenus Sergentomyia were identified. Presence of pointed wing, the small oval hair sockets in the second and subsequent abdominal tergites; the presence of four style spines and the absence of erect hairs on tergal plates were observed in all flies. All female flies had a tubular spermathecae with uniform thickness. All flies had AFi; shorter than AFii+iii and labrum. R2/R 2+3 was less than 0.8.
Identified species
Sergentomyia (Sergentomyia) punjabiensis Sinton:
| • | Phlebotomus minutus var. antennatus Newstead (Sinton, 1924) |
| • | Phlebotomus antennatus Newstead (Sinton, 1927) |
| • | Phlebotomus punjabiensis (Sinton, 1933) |
| • | Sergentomyia (Sergentomyia) punjabiensis (Sinton) (Theodor, 1948) |
| • | Phlebotomus antennatus var. decacanensis (Qutubuddin, 1952) |
| • | Sergentomyia (Sergentomyia) punjabiensis (Sinton) (Lewis 1978) |
Specimen examined (Fig. 2; Table 1,
2)
Male: (n = 5): Abdomen length 540-640 μm; abdomen breadth 260-270
μm; thorax length 360-420 μm; thorax breadth 360-390 μm; head
length 260-280 μm; head breadth 180-270 μm; eye length 130 μm;
eye breadth 90-120 μm; inter ocular 100-110 μm; wing length 990-1470
μm; wing breadth 240-420 μm; halter length 100-140 μm. fore femur:
fore tibia: fore basi tarsi 3.9:3.3:1.7; mid femur: mid tibia: mid basi tarsi
3.8:4.1:2.0; hind femur: hind tibia: hind basi tarsi 3.9:5.0:2.2.
Mouth parts: Labrum is chitinized; bluntly pointed broad structure with three apical sensilla. The hypopharynx is narrower compared to that of the female. Mandible is absent. Maxillary blade has very fine teeth (three). Cibarium is with a concave pigment patch with out any anterior process. Concave arrangement of teeth is found with around 27-30 pointed denticles along with 20-22 small teeth in a single row. Pharynx is broader at base and the constriction is not deep. Pointed teeth are found in the armature.
Labrum length 120-140 μm; maxillary palp length 460-490 μm; palp formula (1,2,3,4,5) with a ratio of 2.83:5.5:7.75:8.5:15.3; proboscis length 150-204 μm; cibarial length 380-400 μm; third antennal segment (Aiii) 70-190 μm; Aiv 45-105 μm; Av 48-105 μm; antennal ascoid/sensilla chaetica on third antennal segment (Aaiii) 20-45 μm; Aaiv 20 μm; Aav 20 μm; ascoid formula is 1/3-15.
| Table 1: | Comparison of the taxonomic characters of female Sergentomyia (Sergentomyia) punjabiensis Sinton (all measurements are in mm) |
| Table 2: | Comparison of the taxonomic characters of male Sergentomyia (Sergentomyia punjabiensis) Sinton (all measurements are in mm) |
Wing venation: First radial vein (R1) 440-660 μm; R2 100-140 μm; R3 180-240 μm; R 2+3 190-380 m; R1 overlap 30 μm.
Genitalia: Coxa is broads at middle. Style has four spines with equal length at apex with accessory setae in middle. Paramere is hooked. Aedegal sheath is thick and slightly curved downwards.
Coxa length 170-230 μm; coxa breadth 30-50 μm; style length 70-90 μm; style breadth 18-20 μm; paramere lobe length 130-200 μm; aedegal sheath length 100-147 μm; genital pump length 80-100 μm; genital filament length 180-350 μm.
Female: (n = 4): Abdomen length 540-640 μm; abdomen breadth 260-270 μm; thorax length 360-420 μm; thorax breadth 360-390 μm; head length 260-280 μm; head breadth 180-270 μm; eye length 120-140 μm eye breadth 100-110 μm; inter ocular 90-120 μm; wing length 990-1440 μm;wing breadth 240-420 μm; halter length 100-140 μm. fore femur: fore tibia: fore basi tarsi 30:23:11; mid femur: mid tibia: mid basi tarsi 32:31:14; hind femur: hind tibia: hind basi tarsi 33:40:18.
| Fig. 2: | Taxonomic feature of Sergentomyia (Sergentomyia) punjabiensis Sinton (Aiv and Aaiv (a), Aiii, Aiv and Av (b), Third palp segment with newstead organ (c), last three antennal segments (d), genital pump (e), hypopharynx (f), pharynx (g), aedegal sheath and paramere (h) and style (i)) |
Mouth parts: Labrum is chitinized, bluntly pointed broad structure with three apical sensilla with 5-6 lateral groups of sensilla. The hypopharynx is broader compared to that of male with a marginal minute serration. Mandible is broad with serrated teeth found along the edge. Maxillary blade has visible teeth (lateral 22-24 and ventral 8). Cibarium is with 26-27 teeth in a concave plate with a pale pigment patch without any anterior process. Chitinous arc is absent. Pharynx is broader at base and the constriction is deep. Pointed teeth are found in the armature.
Labrum length 120-140 μm; maxillary palp length 460-490 μm; palp formula (1,2,3,4,5) with a ratio of 2.9:5.5:7.75:8.5:15.33; proboscis length 110-200 μm; cibarial length 390 μm; third antennal segment (Aiii) 70-140 μm; Aiv 40-70 μm; Av 50-70 μm; antennal ascoid/ sensilla chaetica on third antennal segment ( Aaiii) 15-20 μm; Aaiv 22 μm; Aav 21-22 μm. Pharynx length 150-200 μm; breadth 27-42 μm; armature length 15-60 μm.
Wing venation: First radial vein (R1) 440-660 μm; R2 100-150 μm; R3 180-240 μm; R 2+3 190-380 μm; R1 overlap 30 μm.
Genitalia: Tubular spermathecae with broad anterior end. The individual ducts are thick and fuse to form a short common duct and then fall in to the genital atrium. Spermathecae length is 70 μm and the breadth is 40 μm.
Sergentomyia (Sergentomyia) pondicherriensis (Srinivasan and Jambulingam, 2010): This is a newly described species under the subgenus Sergentomyia by Srinivasan and Jambulingam (2010) from Pillaiyarkuppam village in Puduchery, South India.
Specimen examined (Fig. 3; Table 3,
4)
Male: (n = 2): Abdomen length 550-650 μm; abdomen breadth 260-280
μm; thorax length 330-410 μm; thorax breadth 360-420 μm; head
length 260-270 μm; head breadth 240 μm; eye length 130 μm; eye
breadth 120 μm; inter ocular 110 μm; wing length 1350-1410 μm;
wing breadth 360-420 μm; halter length 140 μm. fore femur: fore tibia:
fore basi tarsi 3.6:3.2:1.8; mid femur: mid tibia: mid basi tarsi 3.8:3.5:1.85;
hind femur: hind tibia: hind basi tarsi 3.9:4.0:2.1.
| Table 3: | Comparison of the taxonomic characters of male Sergentomyia (Sergentomyia pondicherriensis) Srinivasan and Jambulingam (all measurements are in mm) |
| Table 4: | Comparison of the taxonomic characters of female Sergentomyia (Sergentomyia pondicherriensis) Srinivasan and Jambulingam (all measurements are in mm) |
Mouth parts: Labrum is chitinized, blunt, broad structure. The hypopharynx is narrower compared to that of female. Maxillary blade has very fine teeth (2-3). Cibarium is with a pale pigment patch without an anterior process. Concave arrangement of teeth is found in two rows with around 11 in lowest and 8 in the upper rows. Pharynx is broader at base and the constriction is not deep. Pointed teeth are found in the armature.
Labrum length 140-160 μm; maxillary palp length 480 μm; palp formula (1,2,3,4,5) with a ratio of 2.7:5.2:7.55:8.75:16; proboscis length 200 μm; cibarial length 400 μm; third antennal segment (Aiii) 190 μm; Aiv 110 μm; Av 110 μm; antennal ascoid/ sensilla chaetica on third antennal segment (Aaiii) 40 μm; Aaiv 20 μm; Aav20 μm; ascoid formula is 1/3-15.
Wing venation: First radial vein (R1) 440-660 μm; R2 960-1440 μm; R3 180-240 μm; R2+3 190-380 μm; R1overlap 23 μm.
| Fig. 3: | Taxonomic feature of Sergentomyia (Sergentomyia) pondicherriensis Srinivasan and Jambulingam (aedegal sheath and paramere (a), pharynx of female (b), spermathecae (c), wing (d), male cibarium (e), female cibarium (f) and pharynx of female (g)) |
Genitalia: Coxa is broad at middle. Style has four spines with equal length at apex with accessory setae in middle. Paramere is hooked. Aedegal sheath is thick and slightly curved downwards.
Coxa length 200-230 μm; coxa breadth 40-50 μm; style length 70-90 μm; style breadth 20 μm; paramere lobe length 210 μm; aedegal sheath length 120-150 μm; genital pump length 90-100 μm; genital filament length 310-350 μm.
Female: (n = 4): Abdomen length 740-760 μm; abdomen breadth 250-270 μm; thorax length 340-380 μm; thorax breadth 360-400 μm; head length 270-300 μm; head breadth 220-245 μm; eye length 140-150 μm; eye breadth 100-110 μm; inter ocular 100 μm; wing length 1290-1360 μm; wing breadth 300-320 μm; halter length 135-140 μm. fore femur: fore tibia: fore basi tarsi 3.8:4:1.8; mid femur: mid tibia: mid basi tarsi 3.9:4.3:2.0; hind femur: hind tibia: hind basi tarsi 3.4.7.0:2.1.
Mouth parts: Labrum is chitinized bluntly pointed broad structure with three apical sensilla with 5-6 lateral groups of sensilla. The hypopharynx is broader compared to that of male with a marginal minute serration. Mandible is broad with serrated teeth found along the edge. Maxillary blade has visible teeth. Cibarium is with 20-22 teeth in a concave plate with a semicircular pigment patch without any anterior process. Pharynx is broader (barrel shaped) at base and the constriction is not deep. Pointed teeth are found in the armature with a straight line at the posterior.
Labrum length 130-140 μm; maxillary palp length 600 μm; palp formula (1,2,3,4,5) with a ratio of 9.8:18:21:25:48.5; proboscis length 200 μm; cibarial length 450 μm; third antennal segment (Aiii) 70-140 μm; Aiv 40-70 μm; Av 50-70 μm; antennal ascoid/sensilla chaetica on third antennal segment (Aaiii) 15-20 μm; Aaiv 20 μm; Aav 20 μm.
Wing venation: First radial vein (R1) 540-550 μm; R2 170-200 μm; R3 290 μm; R 2+3 250 μm; R1 overlap 20-70 μm.
Genitalia: Tubular spermathecae with broad anterior end and a smooth wall. The individual ducts are with striations and fuse to form a short common duct and then fall in to the genital atrium. Spermathecae length is 70 μm and the breadth is 40 μm.
Sergentomyia (Sergentomyia) dentata Sinton
Specimen examined (Fig. 4; Table 5,
6)
Male: (n=2): Abdomen length 660-680 μm; abdomen breadth 330-340
μm; thorax length 460-500 μm; thorax breadth 360-370 μm; head
length 290-320 μm; head breadth 220-230 μm; eye length 150 μm;
eye breadth 100 μm; inter ocular 100 μm; wing length 1150-1160 μm;
wing breadth 250 μm; halter length 190 μm. fore femur: fore tibia:
fore basi tarsi 13:10:6; mid femur: mid tibia: mid basi tarsi 13.5:13:7; hind
femur: hind tibia: hind basi tarsi 15:17:9.5.
Mouth parts: Labrum is chitinized, blunt, broad structure. The hypopharynx is narrower compared to that of female with bristle like serration (soft). Maxillary blade has very fine teeth (2-3). Cibarium is with a dull pigment patch. Convex arrangement of teeth is with larger (2 μm) laterals (5-6) and smaller minute central teeth (1 μm). Pharynx is broader at base without constriction. Pharynx armature is with scales and ridges.
Labrum length 105-110 μm; maxillary palp length 480-510 μm; palp formula (1,2,3,4,5) with a ratio of 2:5:8:8.5:17; proboscis length 160 μm; cibarial length 25 μm; third antennal segment (Aiii) 75 μm; Aiv 55 μm; Av 55 μm; antennal ascoid/sensilla chaetica on third antennal segment (Aaiii) 30 μm; Aaiv 20 μm; Aav 20 μm; ascoid formula is 1/3-15.
| Fig. 4: | Taxonomic feature of Sergentomyia (Sergentomyia) dentata Sinton (wing (a), female pharynx (b), Aiii, Aiv and Av, Palp segments (c), Aiv and Aaiv (d), genital pump (e), style (f), male pharynx (g), spermathecae (h) and female cibarium (i)) |
| Table 5: | Comparison of the taxonomic characters of female Sergentomyia (Sergentomyia) dentata Sinton (all measurements are in mm) |
| Table 6: | Comparison of the taxonomic characters of male Sergentomyia (Sergentomyia) dentata Sinton (all measurements are in mm) |
Wing venation: First radial vein (R1) 450-470 μm; R2 120 μm; R3 240-260 μm; R 2+3 160-170 μm; R1 overlap is not found.
Genitalia: Coxa is broad from base and tapper. Style has four spines with equal length, with two sub apical and two at apex with accessory setae in middle. Paramere is hooked. Aedegal sheath is thick and slightly curved downwards.
Coxa length 180 μm; coxa breadth 508 μm; style length 60 μm; style breadth 20 μm; paramere lobe length 15 μm; aedegal sheath length 120-125 μm; genital pump length 70 μm; genital filament length 300 μm.
Female: (n = 2)
Abdomen length: 880-890 μm; abdomen breadth 360 μm; thorax
length 460-500 μm; thorax breadth 490 μm; head length 250-270 μm;
head breadth 200 μm; eye length 150 μm; eye breadth 100-110 μm;
inter ocular 115 μm; wing length 1280-1290 μm; wing breadth 300-310
μm; halter length 150 μm. fore femur: fore tibia: fore basi tarsi
4.1:4:4.1.9; mid femur: mid tibia: mid basi tarsi 4.0:4.7:2.0; hind femur: hind
tibia: hind basi tarsi 4.1.4.7:2.1.
Mouth parts: Labrum is chitinized. The hypopharynx is broader compared to that of male with a marginal serration. Mandible is broad with serrated teeth found along the edge. Maxillary blade has visible teeth (16 lateral and 10 ventral). Cibarium is with sharp teeth in a convex plate with larger lateral and shorter central teeth. The pigment patch is semicircular. Pharynx is broader (barrel shaped) at base and the constriction is not deep. Pointed teeth are found in the armature with a convex at posterior.
Labrum length 700 μm; maxillary palp length 540 μm; palp formula (1,2,3,4,5) with a ratio of 14:24:29:33:80; proboscis length 900 μm; cibarial length 400 μm; third antennal segment (Aiii) 70-80 μm; Aiv 40-50 μm; Av 50 μm; antennal ascoid/sensilla chaetica on third antennal segment (Aaiii) 20 μm; Aaiv 20 μm; Aav 25-30 μm. Newstead organ is 20 μm in length and it is found at 20 μm from the 2-3 junction of the palp.
Wing venation: First radial vein (R1) 550 μm; R2 140-150 μm; R3 240 μm; R 2+3 230 μm; R1 overlap 50 μm.
Genitalia: Tubular spermathecae with broad anterior end and a smooth wall. The individual ducts are with striations and fuse to form a short common duct and then fall in to the genital atrium. Spermathecae length is 50-60 μm and the breadth is 30 μm. The length of cerci 170 μm; length of surstyle 77 μm.
DISCUSSION
Seven species have been described under the subgenus Sergentomyia. They are Se. (Ser.) punjabiensis (Sinton), Se. (Ser.) dentata (Sinton), Se. (Ser.) theodori (Parrot), Se. (Ser.) murgabiensis (PerÞliew), Se. (Ser.) mervynae Pringle, Se. (Ser.) fallax afghanica Artemiev and Se. (Ser.) pondicherriensis Srinivasan and Jambulingam, 2010. This is the recent taxonomic report on Sergentomyia species in Sri Lanka after Lewis (1978) apart from Ozbel et al. (2011) where they have stated the presence of some Sergentomyia species with out morphological description. Among these, only Sergentomyia (Sergentomyia) punjabiensis (Sinton) was reported previously from Sri Lanka (Lewis, 1978). This is the first report for the presence of Se. (Ser.) pondicherriensis (Srinivasan and Jambulingam, 2010) and Se. (Ser.) dentata Sinton from Sri Lanka.
Generally, the sandfly taxonomy is based on morphometric, either measurable or countable, characters. In the present study, taxonomic characters such as wing indices, antennal ascoid lengths, lengths of labrum and antennal segments, cibarial dimension and number of teeth along with the pharyngeal dimension and armature type were analyzed. Apart from these, all measurable characters are illustrated in tables to describe the morphology of the Sri Lankan flies precisely for future research.
The subgenus Sergentomyia is extensively studied in some south-east Asian region notably in Pakistan (Kakarsulemankhel, 2004a-c, 2009). The present investigation suggests that there are some minor variations in the taxonomic characters of the Sri Lankan specimens identified as Se. (Ser.) pondicherriensis compared to the other literatures. Males are comparatively larger than the Indian flies (Srinivasan and Jambulingam, 2010), where the females are smaller in size. Third antennal segment (Aiii) is comparatively larger in both male and female flies collected in Sri Lanka. The female flies’ mouthparts and wings are comparatively smaller while the wings are larger in males. The palp formula of female varies. It is 1,2,3,4 and 5 in the present study, whereas it was 2, (1, 3), 4, 5 in Indian flies (Srinivasan and Jambulingam, 2010).
Males and females of Sri Lankan Sergentomyia (Sergentomyia) punjabiensis Sinton have similar taxonomic features compared to the existing literatures from Pakistan (Lewis, 1978; Kakarsulemankhel, 2004a, 2009) except having a smaller wing and larger Aiii.
Male Sergentomyia (Sergentomyia) dentata show similarity with the existing literatures (Kakarsulemankhel, 2004b, 2009; Pringle, 1953; Perfiliev, 1933, 1960, 1968, Sinton, 1933). But they are comparatively shorter. Females also show similarity except for smaller mouthparts. In general the morphometry of the Sri Lankan sandflies belong to this subgenus is found to be similar to previous reports from other countries.
CONCLUSION
The present study provides an evidence for the presence of a rich diversity of the subgenus Sergentomyia of the genus Sergentomyia in the CL endemic localities of Sri Lanka. As the reports from various parts of the world claim the role of this genus in disease transmission, there is a need to study the bionomics, especially the role in leishmaniasis transmission in the country. The present study suggest for an intensive sandfly survey in Sri Lanka to characterize the sandflies morphologically and molecularly as there is an urgent need to identify vectors as one can perceive a role of more than one species in the disease transmission.
ACKNOWLEDGMENTS
The authors wish to acknowledge Mr.Zudardeen, Public health Inspector of Padavi Siripura, Trincomalee. The study received financial assistance from the National Research Council of Sri Lanka (NRC/6/24) and the University Grants Commission of Sri Lanka. The approval from the Department of Wildlife Conservation of Sri Lanka for conducting the study is greatly acknowledged.