J Arthropod-Borne Dis, March 2017, 11(1): 139–146 KE Benallal et al.: Presence of Phlebotomus … 139 http://jad.tums.ac.ir Published Online: March 14, 2017 Original Article Presence of Phlebotomus perniciosus Atypical Form in Algeria *Kamel Eddine Benallal 1, Razika Benikhlef 1, Rafik Garni 1, Brahim Gassen 2, Jean-Pierre Dedet 3, Zoubir Harrat 1 1Laboratoire d’Eco-Epidémiologie Parasitaire et Génétique des Populations, Institut Pasteur d’Algérie, Algeria 2Direction de la Santé Publique de Tamanrasset, Algeria 3Université Montpellier 1 et CHRU de Montpellier, Centre National de Référence des Leishmanioses, Département de Parasitologie-Mycologie, France (Received 2 Nov 2014; accepted 23 Apr 2016) Abstract Background: Phlebotomus perniciosus and Phlebotomus longicuspis are two phlebotomine sand fly species mor- phologically similar and differing in males only by the shape of the copulatory valves which are bifurcated in P. perniciosus, tip long and tapered in P. longicuspis. Methods: A count of the median coxite setae was carried out on 208 specimens from the collections of Dedet and of Parrot, identified previously as P. longicuspis and on 38 P. perniciosus male sand flies captured during the year 2012–2013, in order to seek the presence of atypical P. perniciosus form. Results: The analysis revealed the presence of 33/246 (13%) atypical P. perniciosus previously confused with P. longicuspis species and whose distribution is mainly located in the semi-arid and arid bioclimatic regions. Conclusion: This study proved for the first time the presence of atypical form of P. perniciosus in Algeria. Keywords: Phlebotomus perniciosus, Phlebotomus longicuspis, Atypical phlebotomus perniciosus, Leishmaniasis, Algeria Introduction In Algeria, the first sand fly specimens were reported by Foley and Leduc (1912). Since then, several taxonomic studies were con- ducted on different species and how they might influence transmission of leishmania- sis in Algeria. The recent entomological sur- veys have reported the presence of Phleboto- mus (Transphlebotomus) mascittii Grassi, 1908, P. (Larroussius) chadlii females in the north- east and P. (Paraphlebotomus) kazeruni in the extreme south of Algeria (Berdane-Brouk et al. 2011, Benallal et al. 2013). In the Medi- terranean basin, Phlebotomus (Larroussius) perniciosus Newstead, 1911 is the most com- mon phlebotomine sand fly, related to humid and arid bioclimatic stages. Its distribution spreads out up to the Saharan edge of Tassili and Hoggar, with higher abundance in the humid areas (Rioux et al. 1967, Dedet et al. 1984, Berdjane-Brouk et al. 2012). In Algeria, it is the main vector of Leish- mania infantum MON-1, the parasite that causes the majority of the visceral leishman- iasis (VL) cases in humans and dogs (Izri et al. 1990). This species is often collected in sympatry with a closely related species, Ph. (Larroussius) longicuspis Nitzulescu, 1930. Parrot considered P. longicuspis as a poten- tial vector of L. infantum in the arid areas (Parrot et al. 1941) and recently, this species was found naturally infected with this para- site (Berdjane-Brouk et al. 2012). Females of the Larroussius subgenus are differentiated mainly by the aspect and the dilatation of the *Corresponding author: Dr Kamel Eddine Benallal, E-mail: benallalkamel4@yahoo.fr J Arthropod-Borne Dis, March 2017, 11(1): 139–146 KE Benallal et al.: Presence of Phlebotomus … 140 http://jad.tums.ac.ir Published Online: March 14, 2017 distal spermathecal ducts (Léger et al. 1983, Killick-Kendrick et al. 1990). However, males are distinguished by the morphology of the copulatory valves (aedeagus), with bifurcat- ed apex in P. perniciosus (PN) (Fig. 1B) and with a long, slightly curved, single pointed apex in P. longicuspis (LC) (Fig. 1C) (Parrot 1936 a and b). Previous studies on P. perniciosus males based on the number of median coxite setae highlighted atypical forms of P. perniciosus (PNA) confused with P. longicuspis mainly in Spain (Morrillas-Marquez et al. 1991, Col- lantes and Martinez-Ortega 1997, Martinez- Sanchez et al. 2000), in Morocco (Benabdennbi and Pesson 1998, Pesson et al. 2004, Guernaoui et al. 2006, Boussa et al. 2008) and recently in Tunisia (Ghrab et al. 2006, Boudabous et al. 2008). Then isoenzyme analysis has per- mitted distinguishing these morphs at hexo- kinase (HK) locus thus as molecular biology using random amplified polymorphic DNA (RADP) technique. The result of the three techniques has allowed including PNA with P. perniciosus species (Benabdennbi et al. 1999, Martinez-Sanchez et al. 2000, Pesson et al. 2004, Boudabous et al. 2012). Parrot and Durand-Delacre (1947) reported that a P. longicuspis males collected in BeniOunif (Oran Sahara) displayed a copulatory valves more curved than usual, and in 2012, we identified a male of the subgenus Larrous- sius collected in Tamanrasset (extreme south of Algeria) which exhibited the same fea- tures (curved copulatory valves with a single point). This work aimed whether atypical forms of P. perniciosus in Algeria are present using the morphometric technique. Materials and Methods In order to investigate the presence of atypical P. perniciosus males in Algeria, a retrospective study was done on 208 speci- mens previously identified as P. longicuspis belonging to the two collections, of Louis Parrot (specimens collected between 1934 and 1958) and of Jean-Pierre Dedet (specimens collected between 1973 and 1975). In addi- tion, other samples of the same species (P. perniciosus and P. longicuspis) were captured in animal shelters between 2012 and 2013 by sticky traps in different localities of the coun- try (Setif, Oum-Bouaghi, Blida, Ghardaïa and Tamanrasset) and included in the study. Only males of P. perniciosus and P. lon- gicuspis were slide mounted with Canada bal- sam after treatment in NaOH 20% (Abon- nenc 1972). In all cases, examination of cop- ulatory valves morphology following the key of Dedet (1984) was associated to a counting of the median coxite setae using a Motic 210 camera. The details of the examined specimens and their place of trapping according to bi- oclimatic zones are summarized in (Table 1). Morphometric data (number of median cox- ite setae) were compared using an analysis of variance with t test of Student with Excel software version 2007. Results Overall, 246 males belonging to P. perni- ciosus and P. longicuspis were analyzed. Within the 208 males of the two collections previously identified as P. longicuspis and 38 collected in field, 33 sand flies presented curved copulatory valves characteristic of P. perniciosus atypical male (Fig. 1A). The count- ed setae number of these males varied be- tween 10 and 16 setae (Fig. 2). The number of setae allowed classifying the atypical males with P. perniciosus species that pos- sessed between 10 and 19 coxite setae (Fig. 2) and forked copulatory valves (Fig. 1B). However, the number of median coxite setae of P. longicuspis varied between 18 and 32 (Fig. 2) with long, slightly curved, single point (Fig. 1C). The number of specimens J Arthropod-Borne Dis, March 2017, 11(1): 139–146 KE Benallal et al.: Presence of Phlebotomus … 141 http://jad.tums.ac.ir Published Online: March 14, 2017 examined and their setae mean numbers (+/- Standard Deviation) are summarized in (Ta- ble 2) with the minimal and maximal num- bers of setae. The Student’s t-test showed significant differentiation between P. perni- ciosus and P. longicuspis populations (P< 0.05) (Table 2). The distribution of PNA form is very marked in northwest part of Algeria (Tlemcen and Sidi Bel-Abbes districts) and remained scarce in the rest of regions where- as the abundance of P. perniciosus decreased once moved southward over the country (Fig. 3). Table 1. Sand flies origin and numbers of specimens studied, according to bioclimatic stages Bioclimatic zone Region (n) Collection site GPS coordinate Tizi Ouzou* 30 36°32'20.88"N 4°26'28.55"E Humid Blida (C) 4 36°33'22.45"N 3° 3'17.06"E Blida** 7 / Béjaïa* 7 36°37'40.21"N 5°20'42.62"E Subhumid Boumerdes* 5 36°43'47.24"N 3°32'17.61"E Alger** 35 / Sétif (C) 26 35°41'27.74"N 5°25'43.75"E Arid El Bayadh* 1 33°10'3.76"N 0°28'27.47"E Naâma* 6 32°29'31.70"N 0°28'4.65"O Ain Defla* 1 36°15'47.17"N 2°12'39.07"E Bouira* 3 36° 8'47.98"N 3°49'58.83"E Oran* 7 35°41'54.90"N 0°38'23.72"O Oum Bouaghi(C) 6 35°52'30.79"N 7° 6'48.95"E Semi-arid Relizane* 3 35°42'59.07"N 0°45'21.03"E Sidi-Bel-Abbes* 3 35°14'25.07"N 0°14'43.24"O Tlemcen* 22 34°38'27.30"N 1°33'41.14"O Tlemcen** 34 / Batna** 20 / Constantine** 1 / Béchar* 1 31°55'33.51"N 1°50'19.16"O Béchar** 7 / Ghardaïa (C) 1 32°28'55.09"N 3°42'3.59"E Saharan Tamanrasset (C) 1 22°53'23.07"N 5°21'33.07"E Tamanrasset** 5 / Biskra* 4 35°10'48.41"N 6° 0'32.26"E Biskra** 6 / Total 246 (*) Collection of Dedet, (**) Collection of Parrot, (C) Collected, (/) Data not available and (n) Sample size. J Arthropod-Borne Dis, March 2017, 11(1): 139–146 KE Benallal et al.: Presence of Phlebotomus … 142 http://jad.tums.ac.ir Published Online: March 14, 2017 Table 2. Comparison of the average numbers of median coxite setae by Student test “t” of Phlebotomus perniciosus and Phlebotomus longicuspis Species Number of measure Setae number Min Max P. perniciosus 96 13.67±3.39 10 18 P. longicuspis 150 24.33±5 18 32 Student’ s test 20.69 ddl= 13 P= 0.000 1385 Fig. 1. Copulatory valves of Phlebotomus perniciosus and Phlebotomus longicuspis. A) atypical form of Phleboto- mus perniciosus, B) typical form of Phlebotomus perniciosus, C) typical form of Phlebotomus longicuspis, 1) curved form of copulatory valves, 2) forked form and 3) slightly curved, single point. Bar = 10 µ m Fig. 2. Frequency of median coxite setae for Phlebotomus perniciosus atypical (PNA), Phlebotomus perniciosus (PN) and (LC) Phlebotomus longicuspis. J Arthropod-Borne Dis, March 2017, 11(1): 139–146 KE Benallal et al.: Presence of Phlebotomus … 143 http://jad.tums.ac.ir Published Online: March 14, 2017 Fig. 3. Distribution map of Phlebotomus perniciosus and Phlebotomus longicuspis in Algeria. LC: P. longicuspis, PN: P. perniciosus, PNA: atypical P. perniciosus. Discussion The two collections conserved in Pasteur Institute of Algeria, collection of Parrot which counts 114 species distributed over 16,482 slides, most species belong to the African continent (164 species from 16 countries), to the European continent (36 species from 08 countries) and to the Middle East and Asia (29 species from nine countries), and that of Dedet which counts 4,689 slides with 15 species originating only from Algeria. Despite the small number of specimens re- viewed due to the improper mounting of some specimens which did not allow performing a correct counting of the median coxite setae our results provide, for the first time, the evidence for atypical forms of P. perniciosus in Algeria and thus correct the identification of several specimens identified as P. longi- cuspis. The number of median coxite setae re- mains a powerful tool in the morphological identification since it was previously used to distinguish between P. ariasi/ P. chadlii and P. neglectus/ P. syriacus of Larroussius sub- genus (Benabdennbi and Pesson 1998). It has allowed the description of P. perniciosus atypical male form in Morocco, Spain and Tunisia (Morrillas-Marquez et al. 1991, Benab- dennbi and Pesson 1998, Ghrab et al. 2006). The bioclimatic and geographic repartition of the different analysed sand flies highlighted a different distribution between PN and PNA forms. However, PN are more abundant in the north and northeast of Algeria. This form occupies humid and sub humid bioclimatic zones in concordance with the previous en- tomological surveys conducted in Mila and Jijel by Berchi et al. (2007). Nevertheless the abundance of PNA is rather from the north- west to the great south, thus occupying semi arid, arid and Saharan bioclimatic zones. In Morocco, PNA repartition spreads out from northeast to the south at the Morocco- J Arthropod-Borne Dis, March 2017, 11(1): 139–146 KE Benallal et al.: Presence of Phlebotomus … 144 http://jad.tums.ac.ir Published Online: March 14, 2017 Algerian coast (Boussa et al. 2008), whereas in Tunisia its distribution is higher to south in the arid and semi arid areas (Boudabous et al. 2012). Consequently, in Maghreb the rep- artition of PNA obeys to the same bio- climatic and geographic conditions and seems to be linked to arid climate. Further investi- gations in Libya where distribution of P. per- niciosus is limited in North Africa (Benab- dennbi and Pesson 1998) should give more details about P. perniciosus morphs distribu- tion. The repartition of P. longicuspis is large and this species is found from the north up to Tamanrasset in the extreme south of Algeria. Recently, its distribution area has won more ground since it has been reported also in Burki- na Faso (Depaquit et al. 2005). Nonetheless, LC density remains very low comparing to P. perniciosus and it is found in humid to Saharan bioclimatic stages (Dedet et al. 1984). Conclusion Association of median coxite setae to the standard morphological criteria revealed the presence of 33/246 (13%) of atypical P. per- niciosus until now confused with P. longi- cuspis species mainly located in the semi arid and arid areas. The morphometric tool allowed us to draw a new distribution map of PN, PNA and LC in Algeria and to add the atypical form of P. perniciosus into the list of Algerian phlebotomine fauna. Further bio- chemical, molecular and morphometric stud- ies should be done in order to highlight the status of the different P. perniciosus morphs of Algeria, and also it is important to check the competence of PNA females to develop and to transmit Leishmania sp parasite. Acknowledgements We would like to thank Dr Phillip Scott and Mr Lazreg Sid-Ahmed for English cor- rections. The authors declare that there is no conflict of interest. References Abonnenc E (1972) Les phlébotomes de la ré- gion Ethiopienne (Diptera: Psychodi- dea). Mémoire ORSTOM No 55, Paris. Benabdennbi I, Pesson B (1998) À propos de la variabilité morphologique de Phle- botomus perniciosus (Diptera: Psycho- didae). Bull Soc Franc Parasitol. 16: 53–60. Benabdennbi I, Pesson B, Cadi-Soussi M, Morillas-Marquez F (1999) Morpholo- gical and Isoenzymatic Differenciation of Sympatric Populations of Phleboto- mus perniciosus and Phlebotomus lon- gicuspis (Diptera: Psychodidae) in Nor- thrn Morocco. J Med Entomol. 36(1): 116–120. Benallal K, Gassen B, Bouiba L, Depaquit J, Harrat Z (2013) Entomological inves- tigation following the resurgence of human visceral leishmaniasis in south- ern Algeria. Acta Trop. 128: 518–521. Berchi S, Bounamous A, Louadi K, Pesson B (2007) Différenciation morphologique de deux espèces sympatriques: Phle- botomus perniciosus Newstead 1911 et Phlebotomus longicuspis Nitzulescu 1930 (Diptera: Psychodidae). Ann Soc Ento- mol Franc. 43(2): 201–203. Berdjane-Brouk Z, Charrel R.N, Bitam I, Hamrioui B, Izri A (2011) Record of Phlebotomus (Transphlebotomus) mas- cittii Grassi, 1908 and Phlebotomus (Lar- roussius) chadlii Rioux, Juminer and Gibily, 1966 female in Algeria. Para- site. 18: 337–339. Berdjane-Brouk Z, Charrel RN, Hamrioui B, Izri A (2012) First detection of Leish- mania infantum DNA in Phlebotomus longicuspis Nitzulescu, 1930 from vis- ceral leishmaniasis endemic focus in Algeria. Parasitol Res. 111: 419–422. J Arthropod-Borne Dis, March 2017, 11(1): 139–146 KE Benallal et al.: Presence of Phlebotomus … 145 http://jad.tums.ac.ir Published Online: March 14, 2017 Boudabous R, Amor S, Khayech F, Marzouk M, Bdira S, Mezhoud H, Azaiez R, Sfar M, Babba H (2009) The Phlebotomine fauna (Diptera: Psychodidae) of the Eastern Coast of Tunisia. J Med Ento- mol. 46(1): 1–8. Boudabous R, Jaouadi K, Bounamous A, Babba H (2012) Morphological and molecular investigation of population structure of Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera: Psychodidae) in Tunisia. J Med Ento- mol. 49: 787–793. Boussa S, Boumezzough A, Remy PE, Glasser N, Pesson B (2008) Morpho- logical and isoenzymatic differentiation of Phlebotomus perniciosus and Phle - botomus longicuspis (Diptera: Psycho- didae) in Southern Morocco. Acta Trop. 106(3): 184–189. Collantes F, Martinez-Ortega E (1997) Sobre la validez taxonomia de Phlebotomus lon- gicuspis Nitulescu, 1931 (Diptera: Psy- chodidae). Bol Aso Espanola Entomol. 21: 141–146. Dedet J.P, Addadi K, Belazzoug S (1984) Les phlébotomes (Diptera: Psychodidae) d’Algérie. Cah ORSTOM, Sér Ent Méd Paradol XXI-I. 2: 99–127. Depaquit J, Muller F, Gantier JC, Leger N, Ferte H, Ready P, Niang AA (2005) Phlebotomine sand flies from Ouaga- dougou, Burkina Faso: first record of Phlebotomus (Larroussius) longicuspis south of the Sahara. Med Vet Entomol. 19: 322–325. Foley H, Leduc H (1912) Phlébotomes dans le sud-oranais. Accident simplement locaux dûs à leurs piqûres. Bull Soc Pathol Exot. 5: 511–513. Ghrab J, Rhim A, Bach-Hamba D, Chahed MK, Aoun K, Nouira S, Bouratbine A (2006) Phlebotominae (Diptera: Psy- chodidae) of human leishmaniasis sites in Tunisia. Parasite. 13: 23–33. Guernaoui S, Pesson B, Boumezzough A, Pichon G (2005) Distribution of phle - botomine sand flies, of the subgenus Larroussius, in Morocco. Med Vet En- tomol. 19: 111–115. Izri MA, Belazzoug S, Boudjebla Y, Dereure J, Pratlong S, Delalbre-Belmonte A, Rioux JA (1990) Leishmania infan- tumMON-1 isolé de Phlebotomus per- niciosus en Kabylie. Ann Parasitol Hum Comp. 65(3): 151–152. Killick-Kendrick R, Tang Y, Killick-Kend - rick M, Sang DK, Sirdar MK, Ke L, Ashford RW, Schorscher J, Johnson RH (1991) The identification of the subgenus Larroussiusby the morphol- ogy of the spermathecal ducts. Parassi- tol. 33 (1): 335–347. Léger N, Pesson B, Madulo-Leblond G, Abonnenc E (1983) Sur la différencia- tion des femelles du sous-genre Lar- roussius Nitzulescu, 1931 (Diptera: Phle- botomidae) de la région méditerranéenne. Ann Parasitol Hum Comp. 58: 611–623. Martinez-Sanchez J, Ramiccia M, Pesson B, Morillas-Marquez F (2000) Genetic polymorphism in sympatric species of the genus Phlebotomus, with special reference to Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera: Psychodidae). Parasite. 7: 247–254. Morrillas-Marquez F, Sanchis-Martin MC, Martin-Sanchez J, Acedo-Sanchez C (1991) On Phlebotomus perniciosus Newstead, 1911 (Diptera : Phlebotomi- dae) in the province of Almeria in southern Spain. Parassitol. 33(1): 437– 444. Parrot L (1936 a) Notes sur les phlébotomes XXI. Sur la valve copulatrice de Phle- botomus perniciosus newstead. Arch Inst Pasteur d’Algérie. 14: 144–147. Parrot L (1936 b) Notes sur les phlébotomes. XX. Sur Phlebotomus langeroni var longicuspis Nitzulescu 1930. Arch Inst Pasteur d’Algérie. 14: 137–143. Parrot L, Donatien A, Plantureux E (1941) J Arthropod-Borne Dis, March 2017, 11(1): 139–146 KE Benallal et al.: Presence of Phlebotomus … 146 http://jad.tums.ac.ir Published Online: March 14, 2017 Sur l’infection naturelle des phlébo- tomes par la leishmaniose générale de l’homme et du chien en Algérie. Arch Inst Pasteur d’Algérie. 19: 209–218. Parrot L, Durand-Delacre R (1947) Notes sur les phlébotomes LVI. Phlébotomes de Béni Ounif de Figuig (Sahara ora- nais). Arch Inst Pasteur d’Algérie. 25: 77–86. Pesson B, Ready JS, Benabdennbi I, Mar- tinez-Sanchez J, Esseghir S, Cadi- Soussi M, Morillas-Marquez F, Ready PD (2004) Sand flies of the Phleboto- mus perniciosus complex: mitochon- drial introgression and a new sibling species of P. longicuspis in the Moroc- can Rif. Med Vet Entomol. 18: 25–37. Rioux JA, Golvan YJ, Croset H, Houin R, Juminer B, Bain O, Tour S (1967) Eco- logie des leishmanioses dans le sud de la France. Les phlébotomes. Echantil- lonnage. Ethologie. Ann Parasitol Hum Comp. 42: 561–603.