George Poinar Jr Æ V. Sarto i Monteys. Introduction

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1 Syst Parasitol (2008) 69:13 21 DOI /s Mermithids (Nematoda: Mermithidae) of biting midges (Diptera: Ceratopogonidae): Heleidomermis cataloniensis n. sp. from Culicoides circumscriptus Kieffer in Spain and a species of Cretacimermis Poinar, 2001 from a ceratopogonid in Burmese amber George Poinar Jr Æ V. Sarto i Monteys Received: 3 December 2006 / Accepted: 16 January 2007 Ó Springer Science+Business Media B.V Abstract Heleidomermis cataloniensis n. sp. (Nematoda: Mermithidae) is described from Culicoides circumscriptus Kieffer (Diptera: Ceratopogonidae) in Spain. Diagnostic characters include prominant elevations with multiple genital papillae on either side of the cloacal opening, only one row of genital papillae on the lateral surface of the tail, the tapering tip of the spicule and a reduced vagina. A male intersex of C. circumscriptus parasitised by H. cataloniensis n. sp. has mouthparts resembling those of the female. Two 100 million year-old fossil specimens of an un-named species of Cretacimermis Poinar, 2001, from an Early Cretaceous Burmese amber biting midge of the genus Leptoconops Skuse, show the antiquity of ceratopogonid-mermithid associations. G. Poinar Jr (&) Department of Zoology, Oregon State University, Corvallis, OR 97331, USA poinarg@science.oregonstate.edu V. Sarto i Monteys Fundació CReSA/Entomologia, Universitat Autònoma de Barcelona, Campus de Bellaterra, edifici CreSA, Bellaterra (Barcelona), Spain Victor.Sarto@cresa.uab.es Introduction Biting midges of the genus Culicoides Latreille are widespread and their bites cause annoyance to man and livestock (Mullen, 2002). Some 26 species of Culicoides have been reported from Catalonia (NE Spain) (Sarto i Monteys & Saiz-Ardanaz, 2003; Ventura et al., 2005). Some are potential vectors of two orbiviruses of equines and ruminants, namely African horse sickness disease (AHS) and bluetongue disease (BT), respectively. The devastating bluetongue disease of ruminants has spread into southern Europe within the past decade as a result of climatic changes, but, in 2006, outbreaks were reported for the first time in northern Europe (Germany, the Netherlands, Belgium and Luxembourg). The virus has been acquired and transmitted by autocthonous species of biting midges in Europe and resulted in the deaths of over a million sheep (Purse et al., 2005). Mermithids are the major group of nematodes that utilise biting midges as their definitive hosts. Over 30 species of the Ceratopogonidae, ranging from the tropics to the cool temperate zone, are parasitised by mermithids (Wirth, 1977). Only a few of these parasites have been identified, nevertheless they are important naturally occurring biological control agents. When techniques are eventually developed for rearing these mermithids under artificial conditions, they could be introduced in farm ponds for controlling biting midges. Studies on the biology of biting midges in Spain revealed the presence of a new mermithid belonging

2 14 Syst Parasitol (2008) 69:13 21 to Heleidomermis Rubstov, The present paper describes this species and discusses the effects this nematode has on its host, including the inducement of intersexes. We also characterise two new specimens of Cretacimermis Poinar, 2001, parasitising a 100 million year-old biting midge in Burmese amber, thus showing that ceratopoginid-mermithid associations have been in existence since the Early Cretaceous. Materials and methods Mermithid specimens for the present study were obtained from infected larvae of Culicoides circumscriptus Kieffer screened from a small pond in the municipality of Caldes de Malavella ( N; E) in Girona Province, Spain. Since the mermithids matured to the adult stage inside the host larvae, they could be obtained directly from the infected hosts. Thus most of the infected last instar ceratopogonid larvae contained adult nematodes and juveniles in the process of moulting to the adult stage. Infected host larvae and free-living adult nematodes were heat killed (708C), preserved in 70% ethanol and processed to glycerine for further study. An infected male C. circumscriptus was collected in a UV light trap (Miniature blacklight trap Model 912, John W. Hock Company, Gainesville, Florida, USA) located 2.5 m above ground level outside an enclosure containing sheep and goats on October 25, 2001 in the town of Dosrius ( N; 2825E) in the Province of Barcelona. The two mermithids that emerged from a specimen of Leptoconops Skuse in Burmese amber were provided by Ron Buckley and came from a mine in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin ( N; E), northern Myanmar (Burma). Fossils obtained from the amber beds where the specimen originated have been assigned to the Upper Albian (c mya) of the Lower Cretaceous (Cruickshank & Ko, 2003). The piece containing the fossil mermithids is deposited in the amber collection of Ron Buckley, Florence, Kentucky, USA. Observations and photographs were made with a Nikon SMZ-10 stereoscopic microscope and Nikon Optiphot optical microscope (with magnifications up to 650 ). In the quantitative portion of the following description, the range of the character is given in parenthesis following the mean value. Mermithidae Braun, 1883 Heleidomermis Rubstov, 1970 Heleidomermis cataloniensis n. sp. Type-host: The biting midge, Culicoides circumscriptus Kieffer (Diptera: Ceratopogonidae). Type-locality: Municipality of Caldes de Malavella ( N; E) in Girona Province, Spain. Type-material: Holotype (male) T-603t deposited in the USDA Nematode Laboratory, Beltsville, Maryland; paratypes in both of the authors collections. Description (Figs. 1 9) Adult. Small, white nematodes with smooth cuticle lacking noticeable cross-fibres in adults; head rounded with 2 anterolateral head papillae in single plane and 4 submedial head papillae arranged in second plane just posterior to lateral papillae; posterior to 4 submedial papillae is constriction followed by circle of 6 additional papillae arranged in dorsal, ventral, ventrolateral and dorsolateral positions (in older specimens, both head and neck papillae may become reduced or rudimentary); mouth shifted ventrally, opens near tips of lateral head papillae and anterior to 4 submedial papillae; amphidial openings spherical, shifted slightly dorsal, usually located posterior to neck papillae; amphidial pouch elliptical; 8 hypodermal cords; vulval opening circular; vulval flap absent; vagina reduced in size, barrel-shaped; vaginal canal straight; spicules short, paired, separate, with distal tip slanted ventrally and proximal region bent dorsally; tail long; genital papillae arranged on ventral, lateral and dorsal surfaces of male tail; only single row of 8 11 genital papillae on lateral surface; 4 6 genital papillae occurred on dorsal surface; genital papillae on ventral surface of tail include pre-cloacal papillae and postcloacal papillae; papillae clusters made it impossible to determine actual number of rows on ventral surface; prominant hypodermal elevations with multiple genital papillae on either side of cloacal opening; pharyngeal tube continues almost to cloaca in male. Post-parasitic juveniles (n = 6). Final moults and mating occurs within host (Fig. 7); characters not differing significantly from adults, except for condition of genital organs; postparasitic cuticles lack tail appendage, often partly residual on adults.

3 Syst Parasitol (2008) 69: Figs. 1 5 Heleidomermis cataloniensis n. sp. 1. Lateral view of head of adult female. 2. Ventral view of head of adult female. 3. Lateral view of vulvar area. 4. Lateral view of head of adult male (holotype). 5. Lateral view of male tail. Scalebars: 1, 2, 4, 5 mm; 3, 10 mm; 5, 9 mm

4 16 Syst Parasitol (2008) 69:13 21 Figs. 6 9 Heleidomermis cataloniensis n. sp. 6. Anterior portion of infected larva of Culicoides circumscriptus showing adult nematodes inside (arrows). 7. Posterior portion of mature male within host larva. Arrow shows spicules. 8. Lateral view of male tail showing spicule with slanted tip and prominant elevations with multiple genital papillae on either side of cloacal opening (arrows). 9. Lateral view of vulval area showing barrow-shaped, small vagina (arrow). Scale-bars: 6, 114 mm; 7, 60 mm; 8, 14 mm; 9, 6 mm

5 Syst Parasitol (2008) 69: Infective-stage juveniles (n = 5). Undergo single moult and develop to infective stages within mother nematode; exit occurs through vulval opening; with thin stylet; length mm and greatest width 9 13 mm. Measurements Female (n = 8). L = 4.2 mm ( mm); greatest diameter = 78 (63 107) mm; distance from head to nerve-ring = 281 ( ) mm; V = 44 (41 51)%; length of vagina = 18 (10 24) mm; width of vagina = 12 (6 16 mm); diameter of amphidial opening = 3.4 (3 4) mm; length of amphidial pouch = 12 (11 14) mm; egg diameter = 28 (24 35) mm. Male (n = 9). L = 2.2 ( ) mm; greatest diameter = 56 (32 113) mm; distance from head to nerve-ring = 182 ( ) mm; length of spicules = 46 (32 68) mm; greatest width of spicule shaft = 6 ( ) mm; length of tail = 150 (95 228) mm; body diameter at cloaca = 49 (32 69) mm; diameter of amphidial opening = 5.3 (3.0 10) mm; length of amphidial pouch = 13 (11 21) mm. Remarks There are three additional species of Heleidomermis: H. vivipara Rubzov, 1970, H. ovipara Rubzov, 1974 and H. magnapapula Poinar & Mullens, 1987 (see Rubzov, 1970, 1974; Poinar & Mullens, 1987). The diagnostic characters of H. cataloniensis n. sp. include the prominant hypodermal elevations with multiple genital papillae on either side of the cloacal opening of the male, the presence of only a single row of genital papillae on the lateral surface of the male tail, the tapered spicule tip and the small vagina. In addition, the ovoviviparous condition of the new species separates it from H. ovipara, which is oviparous. The average length of the spicules of H. ovipara are considerable larger (71 mm) than those of H. cataloniensis (46 mm), and there are three rows of lateral genital papillae on the male tail of H. ovipara but only one row in H. cataloniensis. The females of H. vivipara are considerably wider ( mm) than those of H. cataloniensis ( mm), and the length of the vagina is considerable larger in H. vivipara (60 mm) than in H. cataloniensis (10 24 mm). Also the vulval opening is a transverse slit in H. vivipara, whereas it is circular in H. cataloniensis. The amphidial pouches in both H. vivipara and H. ovipara are larger (17 20 mm) than those of H. cataloniensis (11 14 mm). Furthermore, the infective juveniles of H. vivipara are larger ( mm) than those of H. cataloniensis ( mm). The larger females of the North American H. magnapapula (9 14 mm) serve to distinguish this species from females of H. cataloniensis (3 8 mm). The former species also has a larger vagina (35 65 mm) than H. cataloniensis (10 24 mm). Biological observations Rates of larval parasitism of Culicoides circumscriptus collected from a small pond (Fig. 18) ranged from 7 to 68% over the six week sampling period, with a mean infection rate of 25% (n = 520). Parasitised larvae were recovered from the end of August until mid-october during The mermithids killed the host larvae upon exit and Heleidomermis cataloniensis n. sp. is probably the major biological control agent of C. circumscriptus larvae in Catalonia. Studies to determine the host range of this mermithid and whether it could be established in other populations of biting midges in Spain, as conducted by Mullens & Luhring (1998) on the North American H. magnapapula, are being undertaken. The natural distribution of the parasite is probably assisted by parasitised adult hosts flying or being wind-borne to new locations. The mermithids obviously do not emerge from all parasitised larvae, since some are carried into the adult stage. However, the latter, which can still fly, are sterilised as a result of the infection. Intersex (Figs ) An intersex male of Culicoides circumscriptus (Fig. 15) parasitised by H. cataloniensis n. sp. was previously reported (Sarto i Monteys et al., 2003). This condition has been noted in males of other mermithidparasitised ceratopogonids (Callot, 1959; Smith, 1966; McKeever et al., 1997). Aside from the obvious female-like antennae with eight subglobular segments and five elongate distal segments in the C. circumscriptus male (Fig. 14), the mouthparts are also modified. The labrum-epipharynx bears welldeveloped terminal teeth (Fig. 17), the elongate lacinia are armed with minute teeth and setal elements, the well-developed mandibles contain a series of minute, broad, rounded serrations located subterminally, the hypopharynx bears a series of minute teeth and the maxillary palps contain well-developed palpal sense

6 18 Syst Parasitol (2008) 69:13 21 intersexs of C. circumscriptus are physically able to or even have the desire to take blood is not known. Certainly the parasite would benefit from the added nourishment of a blood meal if its presence caused the host to feed. Fossil mermithids parasitising the Ceratopogonidae Figs Fossil mermithids associated with biting midges in 100 mya Burmese amber. 10. Two specimens of Cretacimermis sp. (arrows) adjacent to a female Leptoconops rossi. Specimens deposited in the amber collection of Ron Buckley, Florence, Kentucky. 11. Tail, within partly shed cuticle, of the larger specimen of Cretacimermis sp. that had emerged from L. rossi. Arrow indciates location of cloacal aperture (located on a different focal plane). 12. Cretacimermis protus emerging from a female Atriculicoides swinhoei in Burmese amber. Specimen deposited in the amber collection of Ron Buckley, Florence, Kentucky. Scale-bars: 10, 260 mm; 11, 14 mm; 12, 500 mm organs (Fig. 16). It is not known whether it would have been possible for this male to have taken a blood meal as a result of these modifications. According to Jobling (1928), the labrum-epipharynx, hypopharynx and mandibles together form a piercing organ for cutting the skin of its victim and this action may be assisted by movements of the lacinia. McKeever et al. (1997) concluded that the mouthparts of mermithid-induced male intersexes of C. stellifer were not modified enough for functional blood feeding. Whether male Cretacimermis Poinar, 2001 was established as a collective genus for mermithids from the Cretaceous that cannot be identified to genus or described due to the obscurity of diagnostic characters (Poinar, 2001). The fossil mermithid, C. protus Poinar & Buckley, 2006 was described from the ceratopogonid Atriculicoides swinhoei (Cockerell) in Burmese amber (Fig. 12). Since that study (Poinar & Buckley, 2006), another mermithid-parasitised biting midge in Burmese amber was obtained (Fig. 10). This amber piece contains two mermithids that exited from a female biting midge belonging to the extinct species Leptoconops rossi Szadziewski. That both mermithids are adjacent to the biting midge and the large partly collapsed abdomen of the fly (and the absence of other insects in the amber piece) is strong evidence that the nematodes emerged from this midge. A small hole at the base of the 5th abdominal ventrite of the midge indicates a possible point of exit for one or both mermithids. In addition, the spicule on one of the mermithids indicates that moulting to the adult stage occurred inside the insect. Only species of Heleidomermis, which are restricted to biting midge hosts, and a few mermithid parasites of the Chironomidae are known to moult to the adult stage inside their insect host. All other mermithids exit as postparasitic juveniles and moult to the adult stage in the environment. Both fossil mermithids are complete; but, since most of the diagnostic characters are obscured, they are described here as Cretacimermis sp., as it is impossible to know whether they are conspecific with the previously described C. protus. Cretacimermis Poinar 2001 Cretacimermis sp. The bodies of both nematodes are darkened from the fossilisation process; their lengths are 2.7 and

7 Syst Parasitol (2008) 69: Figs Intersex male Culicoides circumscriptus parasitised by Heleidomermis cataloniensis n. sp. 13. Head showing well-developed mouthparts. 14. Antennae showing normal female pattern with five elongated terminal segments. 15. Abdomen with an emerging worm. 16. Right maxillary lacinia. Note also well-developed sensory structure on third palpal segment (arrow). 17. Tip of labrum with terminal teeth (arrows). Scale-bars: 13, 14, 53 mm; 15, 260 mm; 17, 8 mm

8 20 Syst Parasitol (2008) 69:13 21 belong to the same lineage, since they all show signs of early moulting, C. protus from Atriculicoides has a ventral mouth and the male of Cretacimermis sp. has a spicule and tail shape similar to extant species of Heleidomermis. Maturation to the adult stage within the host may be a primitive trait in certain mermithid lines, especially those that parasitise dipterous hosts that have relatively short life-cycles and live in transitory habitats. Fig. 18 Pond in the municipality of Caldes de Malavella ( N; E) in Girona Province, where the infected larvae of Culicoides circumscriptus were obtained for the present study 2.5 mm, respectively. The heads and tails of both specimens are tapered; however, the oral aperture is obscured and it is impossible to determine whether the cuticle lacks cross-fibres. In the larger specimen, the tail is exposed and a spicule can barely be seen at a distance of 43 mm from the tip (Fig. 11). The spicule is 12 mm in length and the diameter at the cloacal opening is 16 mm, so either these are small individuals or their bodies shrank after entering the resin. A loose cuticle surrounds the tail tip of the male (Fig. 11). The host possesses characters, such as the shape of the wing, forked M-vein, structure of the antennae and elongate proboscis, which identify it as a member of the family Ceratopogonidae (Diptera). Using the key to fossil ceratopogonids in Burmese amber presented in Szadziewski & Poinar (2005), the specimen was identified as Leptoconops rossi Szadziewski, Extant species of Leptoconops Skuse feed on the blood of mammals, birds and reptiles, and the larvae live in moist and often saline soil in arid locations or in sand along coastal and inland beaches (Szadziewski, 2004). Extant species of this genus are parasitised by mermithids (Whitsel, 1965). The nematodes from Leptoconops are shorter in length (2.7 and 2.5 mm) than the type of C. protus (4.7 mm). However, it is not possible to make a detailed comparison between the mermithids from Atriculicoides Remm and Leptoconops due to the obscurity of many diagnostic characters. It is possible that C. protus, Cretacimermis sp. and Heleidomermis Acknowledgements We thank Brad Mullens for general assistance, Ron Buckley for lending his Burmese amber specimen of Leptoconops rossi with the associated mermithid nematodes, Ryszard Szadziewski for help in interpreting the mouthpart structures of the male intersex and Roberta Poinar for comments on an earlier draft of this paper. We are grateful to the Catalonian Department of Agriculture, Livestock and Fisheries for coordination and financial support of the programme for the surveillance of Culicoides from Catalonia, and the Spanish Ministry of Science and Education for funding the Project AGL Sandra Talavera, Nitu Pagès and Mireia Guixà of the CReSA Entomology Unit assisted with the handling of the parasitised larvae and nematodes. References Callot, J. (1959). Action d un Agamomermis sur les caractères sexuals d un Cératopogonidé. Annales de Parasitologie Humaine et Comparée, 34, Cruickshank, R. D., & Ko, K. (2003). Geology of an amber locality in the Hukawng Valley of northern Myanmar. Journal of Asian Earth Sciences, 21, Jobling, B. (1928). The structure of the head and mouthparts in Culicoides pulicaris, L. (Diptera Nematocera). Bulletin of Entomological Research, 18, McKeever, S., Brickle, D. S., & Hagan, D. V. (1997). Mouthparts, antennae and genitalia of intersex Culicoides stellifer parasitized by mermithid nematodes. Medical and Veterinary Entomology, 11, Mullen, G. R. (2002). Biting midges (Ceratopogonidae). In G. Mullen, & L. Durden (Eds.), Medical and veterinary entomology (pp ). New York: Academic Press. Mullens, B. A., & Luhring, K. A. (1998). Age-dependent parasitism of Culicoides variipennis sonorensis (Diptera: Ceratopogonidae) by Heleidomermis magnapapula (Nematoda: Mermithidae) and considerations for assessing parasite impact. Biological Control, 11, Poinar, G. O., Jr (2001). Nematoda and Nematomorpha. In J. H. Thorp, & A. P. Covich (Eds.), Ecology and classification of North American freshwater invertebrates (2nd ed., pp ). New York: Academic Press. Poinar, G. O., Jr, & Buckley, R. (2006). Nematode (Nematoda: Mermithidae) and hairworm (Nematomorpha: Chordodidae) parasites in Early Cretaceous amber. Journal of Invertebrate Pathology, 93,

9 Syst Parasitol (2008) 69: Poinar, G. O., Jr, & Mullens, B. A. (1987). Heleidomermis magnapapula n. sp. (Mermithidae: Nematoda) parasitizing Culicoides variipennis (Ceratopogonidae: Diptera) in California. Revue de Nematologie, 10, Purse, B. V., Mellor, P. S., Rogers, D. J., Samuel, A. R., Mertens, P. P. C., & Baylis, M. (2005). Climate change and the recent emergence of bluetongue in Europe. Nature Reviews Microbiology, 3, Rubzov, I. A. (1970). [A new species and genus of mermithids from biting midges]. Novye i Maloizvestnye Vidy Foewzy Sibiri, 3, (In Russian). Rubzov, I. A. (1974). [New species of mermithids from midges]. Parasitologiya, 8, (In Russian). Sarto i Monteys, V., & Saiz-Ardanaz, M. (2003). Culicoides midges in Catalonia (Spain), with special reference to likely bluetongue virus vectors. Medical and Veterinary Entomology, 17, Sarto i Monteys, V., Hunt, D. J., & Saiz-Ardanaz, M. (2003). Intersex in Culicoides circumscriptus caused by a parasitic nematode of the genus Heleidomermis from Spain. Revista Ibérica de Parasitología, 63, Smith, W. W. (1966). Mermithid-induced intersexuality in Culicoides stellifer (Coquillett). Mosquito News, 26, Szadziewski, R. (2004). Biting midges (Diptera: Ceratopogonidae) from Burmese amber, Myanmar. Journal of Systematic Palaeontology, 2, Szadziewski, R., & Poinar, G. O., Jr (2005). Additional biting midges (Diptera: Ceratopogonidae) from Burmese amber. Polskie Pismo Entomologiczne, 74, Ventura, D., Pagès, N., & Sarto i Monteys, V. (2005). Citas neuvas e interesantes de Culicoides Latreille, 1809 para Cataluña y la Península Ibérica (Diptera, Nematocera, Ceratopogonidae). Boletín de la Asociación Española de Entomología, 29, Whitsel, R. H. (1965). A new distribution record and incidence of mermithid nematode parasitism for Leptoconops kerteszi (Diptera: Ceratopogonidae). Mosquito News, 25, Wirth, W. W. (1977). Pathogens of Ceratopogonidae (midges). In D. W. Roberts, & M. A. Strand (Eds.), Pathogens of medically important arthropods. Bulletin of the World Health Organization (Supplement 1), 55,