First records of the occurrence of twelve species of Sabethini ( Diptera , Culicidae ) in the state of Paraná , southern Brazil

We report on the first records of Sabethini mosquitoes in the state of Paraná, based on specimens collected in forested environments between 2004 and 2005. In total, 12 species were identified, including 7 species of Wyeomyia Theobald, 1901 and 5 species of Sabethes Robineau-Desvoidy, 1827. Among them, 6 are new records for the southern region of the country, and 6 are new records for the state of Paraná. These results indicate the occurrence of relict species of mosquitoes in Paraná, as they were collected from portions of forests that preserve original characteristics.


Introduction
Sabethini (Diptera, Culicidae) are diurnal mosquitoes found both in forested environments and next to anthropic areas.These mosquitoes occur in various regions of the world but are most abundant in tropical and subtropical climates where high amounts of rainfall favor the formation of aquatic micro-ecosystems known as phytotelmata.Sabethini females lay eggs on phytotelmata and the immature forms of this mosquito develop there (Forattini 2002).
In the Americas there are 9 genera of Sabethini in about 221 species, mostly found in tropical climates: Iso stomyia, Johnbelkinia, Limatus, Phoniomyia, Runchomyia, Sabethes, Shannoniana, Trichoprosopon and Wyeomyia.There, the primitive plant formations, climate and soil type determine the distribution and occurrence of mosquitoes.In Brazil, large areas of equatorial, tropical and temperate climate and vegetation have allowed many species of Sabethini to thrive (Lane andCerqueira 1942, Harbach et al. 2007).
However, since European colonization there have been changes in the original vegetation of the Americas caused by land use and other human activities.For instance, the destruction of forests in the state of Paraná, Brazil, started in 1820 with the cultivation of the yerba mate.The original biota of the Atlantic Forest, including mangroves and salt marshes, and which covered 84% of the state's surface, today represents less than 8% of the area.The Atlantic Forest is currently reduced to disjointed spots, most of them constituting formations secondary stages in different successional stages (Maack 1981, Kronen 1990, Morellato and Haddad 2000).
Studies on the fauna of mosquitoes in regions of Paraná that have remaining forests have demonstrated the existence of populations of Sabethini (Bona and Navarro-Silva 2008, Tissot and Navarro-Silva 2008, Muller at al. 2008, 2012, Guedes and Navarro-Silva 2014).In this study, we report new records from protected and residual forests in the state.

Methods
The study area is within the state of Paraná, which is located in southern Brazil Entomological surveys were conducted between 2004 and 2005 in the forested areas of 18 localities, distributed in 20 municipalities (2 points are in contiguous municipalities), represented by environmental preservation areas and private properties, with the presence of residual forests (Table 1; Fig. 1).
Females of Sabethini were collected inside the forest using the human attraction technique.The mosquitoes were captured using a tube containing Chloroform and by hand nets.Ground-level samples were collected over a transect with 18 focal points separated by 10 m.Each central point was positioned about 30 m from a tree in the forest canopy selected for collection.The collector remained for 20 min at each focal point.Collections in the tree tops were made between 10 m and 15 m from the ground and considered the average height of the dominant trees at each site.To collect in the canopy, various types of platforms were accessed through rope ladders and pulleys.
For each species, we give the known distribution in the Americas and the new records.detailing the collection site: geographical coordinates and altitude above (mean) sea level (a.s.l); data collection by human attraction (col.), date of collection; researched forest stratum (CO = Canopy, Soil = SO); and time interval and the number of specimens collected.The information is supplemented with the species' bionomic data and the specimen number in the entomological collection (DZUP).
Specimens were identified with the help of dichotomous keys by Lane (1953) and Forattini (2002), and the literature describing species of Sabethini.Dr Maria Anice Mureb Sallum (Faculdade de Saúde Pública, Universidade de São Paulo) confirmed our identifications.Voucher materials were deposited in the Padre Jesus Santiago Moure Entomological Collection (Diptera), Departamento de Zoologia, Universidade Federal do Paraná (DZUP).
the Walter Reed Biosystematics Unit (2017) online catalog.To check the originality of the species records in southern Brazil and Paraná, we consulted Lane and Cerqueira (1942), and the lists of entomological collections presented by Marchon-Silva et al. (1996), Hutchings et al. (2005), and Tissot and Navarro-Silva (2008).In addition, the lists compiled by Cardoso et al. (2005Cardoso et al. ( , 2010) ) for Rio Grande do Sul and Müller et al. (2008) for Paraná and Santa Catarina were also consulted.The distribution of mosquito species is presented based on the main natural characteristics of Paraná, according to the Maack (1981) and Wons (1985).

Results
The identification of Sabethini mosquitoes collected in forested environments of Paraná, based on the morphological characteristics of the female, resulted in 12 new records, including 7 species of Wyeomyia Theobald, 1901, and 5 species of Sabethes Robineau-Desvoidy, 1827.Among them, 6 are new records for southern Brazil, and 6 are new records for Paraná.Bionomic data.This species occurs in the Terceiro Planalto Paranaense and is found in preserved forest with original characteristics, which is in the small basin of the Paranapanema River.The region has an annual average air temperature of 21-22 °C and average rainfall of 1,600-1,800 mm annually.Bionomic data.The species was collected in the Terceiro Planalto Paranaense in secondary riparian forest of the Tibagi River, near the mouth of the Iguaçu River.In the tropical zone area, the annual average air temperature ranges between 21 °C and 22 °C, while in the subtropical zone areas, the average annual air temperature varies between 19 °C and 21 °C.The average rainfall ranges from 1,600 mm to 1,800 mm annually.

Wyeomyia
Bionomic data.This species is widely distributed in Paraná, occurring in the Serra do Mar and Segundo and Terceiro Planalto Paranaense and covering 4 different landscape features: Dense Ombrophilous Forest (coastal), Campos Gerais, Mixed Ombrophilous Forest Temperate zone and Seasonal Semideciduous Forest.At the Paraná coast, this species was collected in the hydrographic basin of the Laranjeiras Bay, while in the rest of the state it was found in the Itararé, Cinzas, Laranjinha and Iguaçu river valleys, which are part of the basin of the Paraná River.On the coast, the air temperature ranges between 19 °C and 20 °C and the amount of rainfall ranges between 2,000 mm and 3,000 mm annually.In the areas of the subtropical zone the average annual air temperature varies between 19 °C and 21 °C, while in the forest it rangesbetween 17 °C and 18 °C.In these regions, the average rainfall ranges from 1,100 mm to 1,800 mm annually.In the temperate zone, the average air temperatures are lower, between 15 °C and 16 °C, with rainfall of 1,800 mm to 2,000 mm annually.
Identification.Wy. occulta is characterized by the pronotal lobes covered with dark scales with violet reflexes (Fig. 3I) and the wing with the base of vein M covered by long and large scales (Fig. 3K).It differs from Wy. (Hys.)autocratica Dyar & Knab, 1906 by having the pronotum covered with white scales (Fig. 3J).Bionomic data.This species was detected in the Segundo and Terceiro Planalto Paranaense, where it occurred in Seasonal Semidecidual Forest of humid tropical and subtropical areas.Populations of this mosquito were observed in secondary forests on islands of the Paraná River, as well as in western and southwestern forests of the state that retain primitive characteristics, including the presence of bamboo and conifers.In the tropical zone areas, the annual average air temperature ranges between 21 °C and 22 °C, while in the subtropical zone areas the average annual air temperature varies between 19 °C and 21 °C.In these regions, the average rainfall ranges from 1,100 mm to 1,800 mm annually.

Wyeomyia
Identification.Wy. (Wye.)scotinomus has the wings with the base of vein M covered by long and large scales (Fig. 4B) and differs from Wy. (Wye.)nigritubus Galindo, Carpenter & Trapido, 1951 by having the midtarsus covered with white scales from tarsomere (Ta) II to the base of Ta-V (Fig. 4C).Bionomic data.This species occurred in typical Atlantic Forest along the coast of Paraná, within the hydrographic basin of Laranjeiras Bay, which flows directly into the Atlantic Ocean.In this region, the average annual air temperature varies between 19 °C and 20 °C, and the average rainfall is between 2,000 mm to 3,000 mm.

Wyeomyia (uncertain subgenus) undulata
Bionomic data.This species was collected in the Ter-ceiro Planalto Paranaense in secondary tropical forest areas along the banks of the Paranapanema River and on islands of the Parana River.In this region, the average annual air temperature is between 21 °C and 22 °C and average rainfall varies between 1,100 mm and 1,600 mm annually.
Bionomic data.This species occurs in the Terceiro Planalto Paranaense in gallery forests found along the Iguaçu river basin where the average annual air temperature ranges from 15 °C to 16 °C, and the average rainfall is 1,800 mm to 2,000 mm per year.
Identification.Wy. (Mia.)sabethea is differentiated from other species by having wings with the base of vein M covered by narrow scales, the tergite bearing small basal white scales, and maxillary palpi with 3 times the size of the proboscis.Bionomic data.This species was found in the Primeiro Planalto Paranaense, where it occurred in forested portions of Araucaria in the Iguaçu river basin.In this area the average annual air temperature ranges from 14 °C to 15 °C, and average rainfall ranges from 1,600 mm to 1,800 mm.

Identification.
Wy. (Pho.)fuscipes is characterized by antenna shorter than proboscis and proboscis longer than anterior femur (Fig. 5F).This species also differs from other species by having the clypeus without scales,the occiput pronotal lobes covered with scales with purplegreen reflexes (Fig. 5G), and the abdomen with apical white scales from tergite V to VII and absence on tergite VIII (Fig. 5H).Bionomic data.This species was found coexisting with Wy. (Wyo.)scotinomus in areas of seasonal semidecidual forest of the tropical and humid subtropical zones of the Segundo and Terceiro Planalto Paranaense.It occurred on the islands of the Paraná River and along the Iguaçu River in portions of remnant and secondary forests.In the areas at the tropical zone the annual average air temperature ranges between 21 °C and 22 °C, while in the areas of subtropical zone the average annual air temperature varies between 19 °C and 21 °C.In these regions the average rainfall ranges from 1,100 mm to 1,800 mm.Identification.Wy. serratoria is characterized by wings with the base of vein M covered by long and large scales (Fig. 5J), abdomen with basal white scales from tergite II to VII (Fig. 5K) and midtarsus with white scales from apex of tarsomere (Ta) II to the base of Ta-V (Fig. 5L).

Discussion
New records of Sabethini have been found for southern Brazil and the state of Paraná.With 2 exceptions noted below, the occurrence of most species of the tribe is best known for the Amazon region.Wy. shannoni and Wy.undulata had been previously recorded in state of Rio de Janeiro (Lane andCerqueira 1957, Guimarães andArlé 1984).Consequently, the records of the other species in the Atlantic Forest biome and the southernmost records of South America are new.Among the reported species, Wy. (Wyo.)scotinomus was previously only known from Central America (Costa Rica, Panama), the Caribbean island of Trinidad, and South America (Brazil, Colombia and Ecuador) (WRBU 2017, Navarro et al. 2015).
The other species of Sabethini are distributed in various states of the Amazon, for example Sa. (Sab.)shannoni, described from specimens collected in the Amazon, and Sa.(Sbo.)conditus from Rondônia (Cerqueira 1961, Moses et al. 2000).Similarly, Wy. shannoni and Wy.undulata were also described from specimens collected in Acre and Mato Grosso, respectively, and Wy.occulta has been reported as occurring in the state of Pará (Lane andCerqueira 1957, Del Ponte andCerqueira 1938).
Based on current observations, Wy. (Wyo.)scotinomus and Wy.occulta are now known to occur at the Iguaçu river valley, which is their southernmost record (latitude 25° and 26°, respectively).Additionally, Sa. (Sab.)shannoni and Wy.undulata were collected in the Paranapanema river valley (latitudes 23° and 22°, respectively), and Sa.(Sbo.)conditus in the Tibagi river valley (latitude 23°) were collected in northern Paraná.The record of Wy. shannoni on the coast of Paraná in Brazil expands its geographical distribution further south of previous records from areas of dense forest in the coastal states of Rio de Janeiro and São Paulo (Guimarães et al. 1989(Guimarães et al. , 2000)).
Although researchers are still not sure whether Sabethini mosquitoes can transmit infectious agents to humans, their epidemiological importance is notorious, as they have been found naturally infected with arboviruses (Shope et al. 1961, Hervé et al. 1986).Their potential role in the natural cycle of arboviruses instigates us to try to understand how the transmission of these virus occur in the forest environment and whether it can affect humans.The answer to these questions is dependent on new studies on the behavior these mosquitoes in their natural environment, including isolated relict species in areas of remnant forests.

Figure 2 .
Figure 2. Location of the first records of the species in the state of Paraná, Brazil. A. First records for Southern Brazil.B. First records for the state of Paraná.

Figures 3 .
Figures 3. A-C.Sabethes (Sabethes) shannoni: (A) lateral view; (B) Foretarsus with white scales on apex of tarsomere (Ta) II to base of Ta-IV; (C) Midtarsus with white scales on the base of Ta-II to the apex of Ta-IV.D-G.Sabethes (Sabethoides) conditus: (D) lateral view; (E) ociput with golden-green scales; (F) short upper mesepimeral setae, reaching the mesopostnotum; (G) midtarsus covered with white scales on the anterior surface of Ta-II to Ta-IV.H-K.Wyeomyia occulta: (H) lateral view; (I) dorsal view, with pronotal lobes covered with dark scales with violet reflexes; (J) pronotum covered with white scales; (K) wing with the base of vein M covered by long and large scales.

Figures 4 .
Figures 4. A-C.Wyeomyia (Wyeomyia) scotinomus.(A) lateral view.(B) wing with the base of vein M covered by long and large scales.(C) midtarsus with white scales from tarsomere (Ta) II to the base of Ta-V.D-F.Wyeomyia undulata.(D) lateral view.(E) wing with the base of vein M covered by long and large scales.(F) midtarsus with white scales from Ta-II to the middle of Ta-V.G-J.Sabethes (Sabethoides) glaucodaemon.(G) lateral view.(H) dorsal view, occiput covered with scales with purple reflexes.(I) long upper mesepimeral setae, reaching the mesopostnotum.(J) midtarsus with white scales from apex of Ta-II to the base of Ta-V.

Figures 5 .
Figures 5. A-D.Sabethes (Sabethoides) tridentatus.(A) lateral view.(B) dorsal view, occiput covered with scales with bluish green reflexes.(C) long upper mesepimeral setae, reaching the mesopostnotum.(D) midtarsus with white scales from apex of tarsomere (Ta) II to the base of Ta-V.E-H.Wyeomeyia (Phoniomyia) fuscipes.(E) lateral view.(F) antenna (1) shorter than proboscis (2) and proboscis longer than anterior femur (3).(G) clypeus (1) without scales; and occiput (2) pronotal lobes covered with scales with purple-green reflexes.(H) abdomen lateral view, with presence of apical white scales from tergite V to VII (white arrows) and absence on tergite VIII (white arrow).I-L.Wyeomyia serratoria.(I) lateral view.(J) wing with the base of vein M covered by long and large scales.(K) abdomen lateral view, with presence of basal white scales from tergite II to VII (white arrows) (L) midtarsus with white scales from apex of tarsomere (Ta) II to the base of Ta-V.
Argentina and Brazil.

Table 1 .
Geographical coordinates of the field stations where insect collecting took place in the forested areas of Paraná, between April, 2004 to June 2006.