Description of the last instar larva and new contributions to the knowledge of the pupa of Dasyhelea mediomunda Minaya (Diptera, Culicomorpha, Ceratopogonidae)

The fourth instar larva of Dasyhelea mediomunda Minaya is described for the first time and a complete description of the pupa is provided, through use of phase-contrast microscope and scanning electron microscope. Studied specimens were collected in a pond connected to a small wetland “mallin” on the Patagonian steppe, Chubut province, Argentina.


introDuCtion
Biting midges of the genus Dasyhelea Kieffer are a large and complex group of Ceratopogonidae with diverse morphology and biology, occurring worldwide in a variety of habitats (Waugh and Wirth 1976).Taxonomically, the recognition of the subgenera and/or species groups is still incipient and generally, the subgeneric division has been only sporadically applied to various regional faunas (Díaz et al. 2014).At present there are 70 extant species of Dasyhelea inhabiting the Neotropical region (Borkent 2015), of which 12 belong to the cincta species-group as defined by Waugh and Wirth (1976) and, of this number, three occur in Patagonia.Regarding the immatures of this group, only four species are known: D. bahamensis (John-son) (Ronderos et al. 2003), D. cincta (Coquillett) (Spinelli 1983, Díaz et al. 2009), D. mediomunda Minaya (Minaya 1978) and D. paracincta Wirth (Borkent 1991).The original descriptions of the pupa of D. mediomunda by Minaya (1978) and the one given by Spinelli and Wirth (1984) in the revision of Dasyhelea cincta species group from the Neotropical region are very incomplete.
During a recent entomological survey carried out in the vicinity of Esquel, Chubut, immatures of Dasyhelea cincta species group were collected.
The purpose of this paper is to provide the first description of the last instar larva and a full description of the pupa of D. mediomunda.

Materials anD MetHoDs
Larvae and pupae of D. mediomunda were collected with a pipette from a pond connected to a FLORENTINA DíAz, DANIELLE ANJOS-SANTOS, AMPARO FUNES and MARíA M. RONDEROS wetland "mallin".Specimens were carried to the laboratory, larvae were placed individually in plastic containers with water from the same pond and pupae were isolated in a vial with a drop of water and observed daily until adult emergence.Adults were allowed to harden for 24 hours before being preserved to ensure their pigmentation was complete.Immatures and adults were mounted in Canada balsam following the technique described by Borkent and Spinelli (2007).
For Scanning Electron Microscope (SEM), larvae were prepared following the technique of Ronderos et al. (2000Ronderos et al. ( , 2008)).Ink illustrations were made using a Camera lucida attached to a Nikon Eclipse E200 microscope and photographs were taken with a digital camera Micrometrics SE Premium, through the same microscope.Measurements were taken using Compound Microscope (CM).

DisCussion
The larva of Dasyhelea mediomunda is very similar to that of D. bahamensis by virtue of the short head capsule, the hypostoma bearing the medial portion smooth, the maxillary palpus short and button-like, the lateral arms of epipharynx stout and lacking teeth, and the caudal segment lacking hooklets.However, the larva of D. bahamensis differs by having the palatum with one pair of sensilla campaniformia, the scopae absent; the mandible with three teeth, and the hypostoma having fivesix teeth.The pupa is very similar to those of D. bahamensis and D. cincta slender and annulated respiratory organ, which is twice as long as the total length of pupa.The pupae of D. bahamensis and D. cincta differ by the number of pores of the respiratory organ (D. bahamensis has 6 apical and 17 lateral pores and D. cincta bears 8-9 and 14-16, respectively).
Finally, the immatures of D. mediomunda could be also compared with D. paracincta.In the original description, Borkent (1991) mentioned only the head capsule length of the larva and the coloration and number of pores of the respiratory organ of the pupa (pale yellow with apex darkly pigmented, and 11-13 apical and 12-18 lateral pores respectively).

BionoMiCs
Immatures of Dasyhelea mediomunda were collected together with Culicoides lacustris Ronderos in a pond connected to a wetland ("mallin"), fed by groundwater, with intermittent flooding regime, without fishes in the semi-arid Patagonian Steppe (Epele and Archangelsky 2012).Larvae and pupae were collected in December on a sunny day, between 2:00-2:30 pm, air temperature was 20°C, water temperature was 17°C, pH 7.5.In the laboratory, with a temperature of 18-21°C, two larvae lasted five days to reach to pupal stage.Pupae completed their development in seven days.Larvae were motionless or only exhibited slow movements; pupae exhibited circular, slow abdominal movements.

aCKnoWleDGMents
We are grateful to Nélida Caligaris for technical assistance, to Dr. Pablo Pessacq for help in the collection and rearing in the laboratory, and to Dr. Miguel Archangelsky for critical reading of the manuscript.This work was supported by National Research Council of Argentina (CONICET, Argentina).This is contribution number 108 of the LIESA.
Ronderos et al. (2003) in the original description of the larva and pupa of D. bahamensis, incorrectly mentioned 5 dorsoposteromarginal setae (d.p.m.) and 4 ventroposteromarginal setae (v.p.m.).Díaz et al. (2009) in the redescription of pupa of D. cincta also incorrectly mentioned 4 d.p.m. and 3 v.p.m.A detailed revision of the pupae of D. bahamensis and D. cincta during the present study revealed that the d.p.m. (currently named D-IV) and v.p.m (currently named V-IV) are represented by 3 and 2 setae respectively, and the respiratory organ of both species lacks pedicel.FLORENTINA DíAz, DANIELLE ANJOS-SANTOS, AMPARO FUNES and MARíA M. RONDEROS