The immatures of Bezziachilensis Spinelli & Ronderos, 2001 (Diptera, Ceratopogonidae)

Abstract The fourth instar larva and the pupa of Bezziachilensis Spinelli & Ronderos, 2001 are described for the first time. The immature stages were collected from macrophytes and filamentous algae in streams of the Patagonian steppe, in the provinces of Neuquén and Chubut, Argentina. The described stages were photographed and illustrated with a phase-contrast microscope and scanning electron microscope. Data on the bionomics of the species, new records and tables for characters of the known larvae and pupae of Bezzia Kieffer, 1899 from the Neotropical region are provided.


Introduction
Bezzia Kieffer, 1899, a worldwide genus of the tribe Palpomyiini, includes 322 species of which 48 inhabit the Neotropical region, 46 of them recorded by Borkent and Spinelli (2007), and two more recently described: B. ventanensis Spinelli, 2012(Spinelli et al. 2012) and B. galesa Spinelli, 2013(Spinelli et al. 2013. The adults are important predators of small invertebrates and the immature stages are relatively common inhabitants of various kinds of freshwater environments, mainly streams, lakes and ponds, as well as other breeding habitats, such as sphagnum bogs, rice fields, footprints in sandy creek beds, and water gathered in tree holes and bromeliads (Spinelli and Ronderos 2001). The majority of the Neotropical species are known from adults, and only 12 of them are also known as immatures: B. bivittata (Coquillett, 1905), B. blantoni Spinelli & Wirth, 1989, B. brevicornis (Kieffer, 1917; B. bromeliae Spinelli, 1991;B. galesa Spinelli, 2013;B. gibbera (Coquillett, 1905); B. glabra (Coquillett, 1902), B. nobilis (Winnertz, 1852), B. pulchripes Kieffer, 1917;B. roldani Spinelli & Wirth, 1981, B. snowi Lane, 1958and B. ventanensis Spinelli, 2012. Bezzia chilensis Spinelli & Ronderos, 2001 is a member of the venustula species group in the subgenus Homobezzia Macfie, 1932, distributed in Valparaiso Province (Chile), and Salta and Río Negro provinces (Argentina) (Spinelli and Cazorla 2003). During a recent survey carried out in the northwestern Argentine Patagonia, larvae and pupae of B. chilensis were collected. The purpose of this paper is to describe the fourth instar larva and pupa of this species, with phase-contrast and scanning electron microscopy (SEM) and to provide tables for characters of the known larvae and pupae of Bezzia from the Neotropical region.

Material and methods
Larvae and pupae were collected on the bordering vegetation in three streams on the Patagonian steppe in the provinces of Neuquén and Chubut. The substrate was removed with the aid of a strainer and transferred to a white tray where larvae and pupae were collected with a pipette. Further substrate samples were carried to the laboratory to search for more specimens. Larvae were placed in individual containers with water and substrate from their natural environment. Pupae were isolated in a vial with a drop of water, and observed daily until adult emergence. Adults were allowed to harden for 24 h before being preserved in ethanol to ensure their complete pigmentation. For detailed examination with a phase-contrast microscope, larval and pupal exuviae and adults were mounted in Canada balsam following the technique described by Borkent and Spinelli (2007). Mounted larval exuviae were oriented ventral side up to facilitate examination of the epipharyngeal combs within the head capsule. Pupal exuviae were mounted dorsoventrally. Photomicrographs were taken with a Micrometrics SE Premium digital camera, through a Nikon Eclipse E200 microscope and a Leica EC3 digital camera, through a Leica DM 500 microscope. Illustrations were drawn with a camera lucida and Adobe illustrator CC. The map was drawn in QGIS v. 2.14. Larvae were also examined using scanning electron microscopy (SEM) (JOEL 2000) following the technique of Ronderos et al. (2000Ronderos et al. ( , 2008. Measurements were taken with a (BCM) Leitz Wetzlar binocular compound microscope. The temperature of the water and air were measured with an alcohol thermometer in degrees Celsius. For larval terms and abbreviations of measurements, see Anjos-Santos et al. (2017); for pupal terms, see Borkent (2014). Studied specimens are deposited in the collection of the Museo de La Plata, La Plata, Argentina (MLPA).

Bionomics
The immature described here were collected in northwestern Argentine Patagonian steppe (Fig. 5): río Cuyín Manzano, located in the Parque Nacional Nahuel Huapi in southern Neuquén Province and arroyo La Cancha, arroyo Madera and arroyo Montoso, that flow into tributaries of the Chubut river in northern Chubut Province. All sites are surrounded by shrubby steppe, composed mainly of willow tree (Salicaceae) and grass, and are used as a water source for cattle. Immatures were collected in puddles of water with a rocky or sandy bottom, on the bank of the streams, among macrophytes (Ceratophyllum L.), bryophytes and filamentous algae. Larvae were distributed through all the puddles, but pupae only in the bordering vegetation. In La Cancha, Madera and Montoso streams water temperature ranged between 15-19 °C, and air temperature between 16-25 °C. In Cuyín Manzano river, the air and water temperature data were not measured. Under laboratory conditions, the larvae took 4-14 days to reach the pupal stage, and 2-7 days to complete its development to the adult stage. Pupae found at the site completed their development in 1-8 days. Larvae of Bezzia chilensis showed the same movement reported by Spinelli et al. (2013) for B. galesa, alternated fast undulating movements with static periods. Pupae observed on trays showed a semi-circular, slow abdominal movement typical of other ceratopogonid pupae.

Taxonomic discussion
In a series of contributions reviewing the Neotropical Bezzia, Spinelli and Wirth (1989a, b, 1990 recognized the subgenus Bezzia, including the gibbera, nobilis and punctipennis groups, and the subgenus Homobezzia including the dentifemur, glabra, venustula and brevicornis groups. These papers also present diagnoses, descriptions and keys to subgenera and species groups, the last ones based mainly on adult characters. Spinelli et al. (2012) suggested a cladistic analysis is needed to propose a phylogenetic classification of the genus Bezzia. Borkent (2014) presented a generic pupal description of Bezzia and in his taxonomic discussion mentioned the difficulties in diagnosing the genus and affirmed that providing a key to the species in a given region is superfluous. The current knowledge of immature stages of the Neotropical Bezzia is incipient. The subgenus Bezzia has only two species known as larvae and two known as pupae and the subgenus Homobez-  Ronderos et al. (2007) zia has five known as larvae and 10 known as pupae, with some of these immatures being poorly described and impossible to compare with their congenerics. Main diagnostic characters for larvae and pupae are given in the Tables 1 and 2, respectively. The immatures of Bezzia chilensis are herein compared with four species belonging to the subgenus Homobezzia; these four are the only ones which have a complete description: B. blantoni (described by Ronderos and Spinelli 2009), B. galesa (described by Spinelli et al. 2013), B. roldani (described by Ronderos et al. 2007) and B. ventanensis (described by Spinelli et al. 2012).
The larva of Bezzia chilensis shares with B. blantoni, B. galesa and B. roldani features typical of predatory larvae: hooked mandibles with fossa mandibularis, epipharynx less massive with 2 combs and cylindrical maxillary palpus (Hribar and Mullen 1991). The labrum and palatum sensilla are very similar among the species. However, the larvae of these three species can be distinguished from B. chilensis by the features given in Table 1 and by the following additional characters: Bezzia blantoni by the maxilla with a blunt sensillum, epipharynx with 4-6 stout, short teeth and auxiliary sclerite shorter; Bezzia galesa by the maxilla with a blunt sensillum, galeolacinia with a stout, sharp, pointed and medium-sized seta, epipharynx with 6 or 7 stout and small teeth on the ventral comb; and Bezzia roldani by the W-shaped palatal bar and the ventral comb of the epipharynx bearing 4 or 5 stout and short teeth. The larva of B. ventanensis remains unknown.
With regard to the pupa, besides the features given in Table 2 Spinelli et al. (2012) In addition, a detailed revision during this study revealed that D-4-T of B. galesa is a seta and was erroneously described as campaniform sensillum by Spinelli et al. (2013) and D-5-T and D-8-I of B. ventanensis described by Spinelli et al. (2012) as campaniform sensilla are long, a thin seta and a medium-sized seta, respectively.
The pupae of Bezzia chilensis and the other Neotropical known pupae of Bezzia share the features of the generic description given by Borkent (2014). However, we agree on the need of a revision of the genus within a phylogenetic analysis and the redescription of the incompletely described immatures, emphasizing as well the importance of describing immatures for a better knowledge of the genus.