Redescription of immatures of Dasyhelea flavifrons Guérin-Méneville ( Culicomorpha : Ceratopogonidae ) and new contribution to the knowledge of its larval habitats

The fourth instar larva and pupa of Dasyhelea flavifrons Guérin-Méneville are redescribed, illustrated, and photomicrographed using binocular, phase-contrast, and scanning electron microscopy. Comparisons with the American species of the grisea group were made. The immatures were collected by using a siphon bottle in tree-holes and from water collected in dead snail shells in Salta Province, Argentina, transported to the laboratory and there bred to the emergence of the adults. Details on larval habitats are given. These are the first records from Argentina and in gastrotelmata.


INTRODUCTION
The biting midges Dasyhelea Kieffer are common and mainly diurnal dipterans occurring in all zoogeographical regions, except Antarctica.The larvae of Dasyhelea develop mostly in shallow aquatic and semiaquatic habitats, and in general feed on detritus and algae, though a few species feed in dead insects (Borkent and Spinelli 2007).
Adult females feed on nectar from flowers and pollen.Moreover, some are important pollinators of commercial trees such as cacao (Theobroma cacao) and rubber (Hevea brasiliensis) (Borkent andSpinelli 2007, Córdoba et al. 2013).
Taxonomically, the recognition of subgenera and/or groups of species have been proposed for all regions of the world.Borkent (2016) listed 77 species for the Neotropics, of which only 12 are known also from their immatures.Subsequently, grogan et al. (2016) described 13 new species from guadeloupe, and Díaz et al. (2017) described two species from Peru and Brazil, all based on adults.Recently, Díaz et al. (2018) described two species, including their immature, from the Yungas ecoregion, Argentina.
T h e p h y t o t e l m a t a a r e a q u a t i c microenvironments formed by the accumulation of water in any part of the body of plants (e.g.leaves, flowers, stems, trunks, tree holes and artificial containers).The gastrotelmata are aquatic microhabitats formed by the collection of water (usually from rainfall) in the shells of dead snails (Janetzky et al. 1995).In spite of their small size, snail shells retain water longer than any other natural container, providing a relatively permanent habitat (Lounibos 1980).The communities of organisms that live in these microenvironments are simple and based on detritus; the macroinvertebrates most frequently found are insects, immature stages of Diptera being dominant (Campos et al. 2011).The knowledge of Ceratopogonidae from Argentina that breed in these microenvironments is poor; only 7 of the 31 genera registered (Borkent and Spinelli 2007) have been associated with phytothelmata: Atrichopogon Kieffer, Forcipomyia Meigen, Dasyhelea Kieffer, Culiciodes latreille, Stilobezzia Kieffer, Bezzia Kieffer, and Palpomyia Meigen (Campos et al. 2011).
During a sampling program focused on the collection and study of Diptera that was carried out in Salta province between 2011 and 2012, larvae and pupae were collected from tree holes and dead snail shells, and reared to adults.Based on the characters of adult males, they were identified as Dasyhelea flavifrons.This is considered a wide spread Holarctic arboreal species, whose larvae have been reported in europe as terrestrial and inhabiting sap flows, mushrooms and tree holes (Dominiak and Szadziewski 2010).However, the original description and the different redescriptions of immatures are incomplete, except for the contribution of Keilin (1921), who described all stages of this species under the name D. obscura Winnertz, but also incompletely.The aim of this work is to provide the full redescription of these immature in accordance with modern standards and to present the first records from Argentina and in gastrotelmata as larval habitat.

STUDY AREA
San Ramón de la Nueva Orán (hereafter Orán), is a city established in northwestern Argentina near the border with Bolivia (23º08' S, 64º20' W, elevation 337 m).The region has a subtropical climate, with an average summer temperature of 27.7 °C and winter temperature of 16.4 °C, and a mean annual rainfall of 1,000 mm, occurring mostly from October to April, the warmer months.
The study area has been described in more detail in Mangudo et al. (2015).Briefly, Orán is located in the pedemontane floor of the Yungas subtropical montane moist forest (Brown et al. 2001), which remains mostly to the east and North of the city.Besides urbanization, other human related ecological modifications include industrial development, agriculture and forestry (Brown et al. 2001).The city is characterized by a densely built central area surrounded by suburban areas with bigger gardens and more trees; throughout the city most buildings are low.
ENTOMOLOGICAL SAMPLING larvae, larval exuviae and pupae were collected between February and March in 2011 and 2012, as a part of a larger study on mosquito (Diptera: Culicidae) larval habitats.Samples were collected from tree holes using a siphon bottle (Müller andMarcondes 2006, Mangudo et al. 2010) and from snail shells by overturning and examining the water content from each shell in a white plastic tray, from which larvae and pupae were collected with a pipette.Tree holes were located in the city and in yunga forest patches to the north and east of town, while snail shells were collected from forest patches only.For details on tree holes selection see Mangudo et al. (2015Mangudo et al. ( , 2018) ) and for snail shell sampling see Mangudo et al. (2017).larvae were preserved in ethanol 80% and pupae were kept alive in the laboratory isolated in plastic vials (2 mm) holding water from the larval habitat and containing a piece of humid filter paper, to maintain the humidity inside the vials, until adult emergence.emerged adults were maintained alive for 24 hours to ensure the development of their final pigmentation.Adults and their respective exuviae were stored in vials containing 80% ethanol.larval, pupal exuviae and adults were mounted in Canada balsam following the technique described by Borkent and Spinelli (2007).The technique of Ronderos et al. (2000Ronderos et al. ( , 2008) ) was followed to prepare larvae for scanning electron microscopy (SeM).A camera lucida was used to make illustrations with pen and ink.Photomicrographs were taken with a Micrometrics Se Premium digital camera, through a Nikon Eclipse E200 microscope.For larval terms see Díaz et al. (2018) and for pupae see Borkent (2014).
The plates were made in TIFF format in Adobe Photoshop version14.The material studied is deposited in the División entomología, Museo de la Plata (MlPA), la Plata, Argentina.

COMPARISON
Dasyhelea flavifrons belongs to the Dasyhelea grisea species group as defined by Waugh and Wirth (1976), and this group is equivalent to the subgenus Dasyhelea s. str.(Dominiak 2012) and has a wide distribution.Guérin-Méneville in 1833 described for the first time the immatures collected from elm flux.Subsequently, various authors with SEM the immature.The larva of D. flavifrons is very similar in having the mandible with four teeth, the maxillary palpus short and button-like and the caudal segment with anterior ring of small spines.The larva of D. necrophila differs in the labrum with two pairs of sensilla campaniformia; the epypharynx lacking the auxiliary sclerite and the caudal segment with 5-6 pairs of hooks.On the other hand, the pupa of D. necrophila differs by the total length 2.70 mm, the posterior margin of dorsal apotome with two tubercles; the clypeal labrals long and thin; the respiratory organ 30 apical and 4 lateral pores and tergite 1 with l-2-I, L-3-I short seta.The male pupa has the genital lobe not reaching posterior margin of segment 9. Díaz et al. (2013) described the larva and pupa of D. eloyi collected from flooded soil and from water lettuce (Pistia stratiotes l.) and differs in the longer head capsule; the labrum with three pairs of sensillae campaniformia; the scopae with 4-6 teeth; the lateral arms of epypharynx smaller and the caudal segment longer.The pupa of D. eloyi is readily distinguished from D. flavifrons in the following characters: the posterior margin of dorsal apotome with two tubercles and DA-1-H long; the clypeal labral sensilla long and thin; the anteromedial sensillum AM-2-T short; the respiratory organ with 16-18 apical and 6-8 lateral pores, and the D-2-T represented by a peg; segment 4 with l-1-IV medium-sized and segment 9 longer than in D. flavifrons.Díaz et al. (2014) described the pupa of D. pseudopollinosa that was collected together with D. paulistana and can be distinguished from D. flavifrons by its small total length (2.8 mm); the dorsal apotome smooth; the respiratory organ smooth bearing 18-20 apical pores and the segment 9 with terminal process straight, as long as the length of the body of segment 9. The male pupa has the genital lobe reaching the posterior margin of segment 9.
Recently, Díaz et al. (2018) described the immature of D. azteca and D. yunga that were collected from small temporary ponds and stream.The larva of D. flavifrons shares with the larva of D. azteca the following characters: the total length 4-5 mm; the palatum with four pairs of sensillae campaniformia; the galeolacinia with short, stout seta, 5-6 papillae and two flap-like lobes; the maxillary palpus short and button-like; the lateral arms of epipharynx with auxiliary sclerites and hypopharynx with posterior comb with fringe.However, D. azteca differs in having two pairs of sensilla coeloconica; the scopae with 14-16 strong teeth; the mandible with three teeth, and caudal segment with 10-12 hooks.The pupa differs in the smaller total length (2.22 mm); respiratory organ bearing 22-24 apical and 3-4 lateral pores; dorsal apotome with DA-1-H short seta; the clypeal-labral sensilla Cl-1-H medium-sized, thin seta, and Cl-2-H short, thin seta; anterolateral sensilla AL-1-T long, thin seta, AL-2-T medium-sized, thin seta and AL-3-T campaniform sensillum; the respiratory organ with 16-18 apical and 6-8 lateral pores, and D-2-IV short seta.The male pupa has genital lobe extending slightly beyond of posterior margin of segment 9.
Finally, the pupa of D. yunga Díaz differs clearly by its exuviae yellowish, with dorsal apotome smooth and without clypeal/labrals, respiratory organ pale brown, smooth, with 18-20 apical and 3-4 lateral pores and genital lobe of pupa male moderately wide and globose extending beyond posterior margin of segment 9.

BIONOMICS
The tree holes where the larvae and pupae of Dasyhelea flavifrons were collected were pans formed as branch intersections (maintaining an unbroken bark lining) and rot holes (lacking bark lining and penetrating into the wood of the tree).The specimens described herein were collected from urban tree holes in Delonix regia (Bojer) Raf.(Fabales: Fabaceae), and in holes from unidentified trees in yunga forest patches to the east of the city.Other specimens were also collected in the city from Bauhinia sp.(Fabales: Fabaceae), Citrus sinensis Osbeck (Sapindales: Rutaceae), Morus spp.(Rosales: Moraceae), and Thevetia nereifolia Juss.(Gentianales: Apocynaceae).The finding of immature D. flavifrons in tree holes in northwest Argentina is consistent with the described larval habitats in the Holartic region, since larvae and pupae have been usually collected from tree holes and sap flows of various species, such as Quercus, Fagus, Ulmus and Fraxinus (Oboňa and Dominiak 2014; see also revisions on Dominiak andSzadziewski 2010, Dominiak 2012).
This species was found in tree holes either as single species or coexisting with other species of Ceratopogonidae, Culicoides trilineatus Fox, the latter species suspected to be a vector of BTV.The immatures of D. flavifrons were also found coexisting with Aedes aegypti l. (vector of dengue, urban yellow fever, zika, and chikungunya virus (gubler 2004, Ayres 2016, Marcondes et al. 2017)), Haemagogus spegazzini Brèthes (potentially vector of arbovirus (Karabatsos 1985), or Toxorhynchites guadeloupensis Dyar and Knab (Diptera: Culicidae).
In the shell of dead Megalobulimus lorentzianus (Doering) snails, D. flavifrons were found as a single species (specimens described herein).The snail shells width and length of aperture were approximately 3 cm and 5.5 cm and held less than 0.5 ml of water.Other Megabulimus shells held larvae or pupae of the mosquito Limatus durhamii Theobald (Diptera: Culicidae) and/or immature Psychodidae.To our best knowledge, this is the first report of Ceratopogonidae collected from gastrotelmata in Argentina.