INTRODUCTION

Psorophora (Janthinosoma) cyanescens (Coquillett, 1902) is a mosquito native to America. It has been reported in Argentina, Bolivia, Colombia, Guatemala, Guyana, Mexico, Nicaragua, Paraguay, Peru, Trinidad and Tobago, United States, Uruguay, and Venezuela (^{WRBU, 2021}).

Females of Ps. cyanescens are persistent biters on humans and livestock and are rather difficult to detach from the host once they have started to take a blood meal (^{Snow et al., 1960}). The first adults appear about a week after heavy rains and may persist for two or three weeks thereafter (^{Snow et al., 1960}). Although, to our knowledge, no pathogen has been isolated from Ps. cyanescens, its importance lies in the fact that they are annoying and voracious blood biters. In Mexico, pools of Ps. cyanescens were tested for RNA of Flavivirus, Alphavirus, and Orthobunyavirus, and were negative (^{Farfan-Ale et al., 2009}; ²⁰¹⁰). However, its vectorial capacity cannot be ruled out because other members of Psorophora are potential vectors of arboviruses. Rocio and West Nile viruses were isolated from Ps. ferox in Brazil and USA, respectively (^{de Souza Lopes et al., 1981}; ^{Kulasekera et al., 2001}). In Argentina, Western Equine Encephalitis virus was isolated from Psorophora pallescens Edwards, 1922 (^{Mitchell et al., 1987}), and in Colombia, Venezuelan equine encephalitis virus was found in Psorophora confinnis Lynch Arribálzaga, 1891 (^{Hoyos-López et al., 2016}).

Most taxonomic studies on mosquitoes are based on the morphological description of larvae and adults. On the contrary, minor importance has been paid to egg morphology despite than exochorion pattern in mosquitoes has taxonomic value because allowed to identify of the species complex and has been useful in phylogeny studies (^{Bosworth et al., 1983}; ^{Reinert et al., 2009}). Furthermore, the description of mosquito eggs by scanning electron microscopy (SEM) allows more detailed descriptions of the ornamentation of the egg exochorion, and which provide diagnostic characteristics for the species (^{Bosworth et al., 1983}). Currently, in Mexico there is no study that provides data on the morphological patterns of the exochorion of mosquito eggs of even the most important medical and veterinary species.

Genus Psorophora includes 49 species divided into the subgenera Grabhamia (16 species), Janthinosoma (23 species), and Psorophora (10 species) (^{Harbach, 2013}; ^{WRBU, 2021}). Currently, eggs of three species of Janthinosoma, and one of Grabhamia have been described by SEM (^{Bosworth et al., 1983}; ^{Pacheco et al., 2012}, ^{de Mello et al., 2017}; 2018). Currently, no descriptions of the morphological patterns of the exocorion of mosquito eggs have been made in Mexico. Therefore, to contribute to the state of knowledge about the exochorionic pattern of Psorophora, we used the SEM technique to describe Ps. cyanescens eggs and to provide fertility data from females collected in Yucatan, Mexico.

MATERIALS AND METHODS

Study site. The mosquitoes were captured in the forest near the community of Ekmul (20° 57’ 54’’ N, 89° 20’ 56’’ W), located 27 km east of Merida city, the capital of Yucatan state. Yucatan has a distinct rainy (May-October) season and a dry season (November-April). In the rainy season, the mean rainfall is 1,000 mm, and the mean temperature is 27.5 °C. In the dry season, the mean rainfall is 300 mm, and the mean temperature is 25.1 °C (^{INEGI, 2021}).

Mosquito collections. Adults of Psorophora were captured in October 2020 (rainy season) using a backpack-mounted aspirator (Prokopack Aspirator®, model 1419, John W. Hock Company). The mosquitoes were captured in a low deciduous forest. The area is populated by trees typical of the region, such as Alvaradoa amorphoides, Bursera simaruba, Ehretia tinifolia, Havardia albicans, Leucaena leucocephala, Mimosa bahamensis, Piscidia piscipula, and the Bromelia karatas plant. Mosquitoes were captured during flight or when they landed on the entomologist. The capture was made between 16 and 18 hours. Mosquitoes were transported alive to the Laboratorio de Arbovirología at Universidad Autónoma de Yucatán and identified according to species using a published identification key (^{Darsie Jr & Ward, 1981}).

Females of Ps. cyanescens (n = 10) were blood-feed on anesthetized mice (Animal Ethic authorization: CEI082015-CIR-UADY). Females were allowed to develop their eggs; they were individually kept in cardboard boxes at 27 °C and 70% RH and were fed sugar solution (10%) through cotton pads. A plastic container (7 x 7 x 5 cm) filled with 50 ml of de-chlorinated water (Hycel®) was used as the oviposition site.

Scanning Electron Microscopy of eggs of Ps. cyanescens. The SEM methodology was similar to previous publications, with some modifications (^{Pacheco et al., 2012}; ^{de Mello et al., 2017}). Briefly, the eggs were removed from the filter paper using a brush and were fixed in 2% glutaraldehyde and postfixed in 1% cacodylate buffer. Subsequently, the eggs were dehydrated in a series of increasing ethanol concentrations and critical point dried with CO₂. The eggs were then sputter-coated with gold. The SEM micrographs were obtained with a Digital Scanning Microscope (Hitachi VP-SEM SU1510, Hitachi High Technologies America, Inc.) at the Laboratorio Nacional de Biodiversidad, Instituto de Biología, Universidad Nacional Autónoma de México (UNAM).

The morphology of the eggs was described using specialized terminology (^{Harbach & Knight, 1980}). Egg measurements are given in micrometers and expressed as the mean and standard deviation.

RESULTS

Measurement and morphology of eggs of Ps. cyanescens. In general, the eggs of Ps. cyanescens are black, elliptical in shape, and very wide. On average, the eggs measure 734.26 µm (± 33.52) in length and 245 µm (± 12.96) in width, with an egg index (length/width ratio) of 3 ± 0.26 μm (Table 1; Fig. 1). The exochorion is characterized by polygon pattern, where each polygon consists of longitudinal ridges joined by cross-ridges that define regular “hexagonal” areas. Each longitudinal ridge consisting by a small tubercle. The small tubercles are irregular, rectangular, rounded, or tubular, and their densities ranged from 28 to 36 tubercles per cell (Figs. 2A, B). The ornamentation of exochorion also presents long and conical tubercles throughout the egg region. Long tubercles are located at the base of the chorionic cell. The micropylar apparatus located in the anterior region of the egg and has a prominent, continuous and thickness collar (Fig. 2C).

Figure 1 Ventral (upper) view, anterior end at the top of the entire eggs of Psorophora cyanescens. MiC, micropylar collar; OCT, outer chorionic tubercle. 

Figure 2 Egg of Psorophora cyanescens. A) typical ornamentation of the outer chorionic reticulum showing long tubercles (LT) located at the base of the chorionic cell; B) Outer chorionic reticulum (OCT), the shadows indicated by the white arrows indicate the absence of long tubercles; C) anterior region of the egg showing the micropylar apparatus, formed by a micropyle (Mi), micropylar disc (MiD), and micropylar collar (MiC); D) posterior region of the egg showing long tubercles (LT). 

Fecundity of Ps. cyanescens. Psorophora cyanescens laid mature eggs at 60 hrs. post-feeding (2.5 days). Of the 35 females, only ten laid a total of 822 eggs. The mean number of eggs per female was 82.20 (± 13.31).

DISCUSSION

With the contribution of the present work, there are currently five species of the genus Psorophora, to which SEM describes the morphology of the eggs. Previously in the subgenera Janthinosoma, eggs of Psorophora ferox (von Humboldt, 1819), Psorophora albigenu (Lutz, 1908), and Psorophora albipes (Theobald, 1907) were described by SEM (^{Pacheco et al., 2012}; ^{de Mello et al., 2017}; ²⁰¹⁸). While in the subgenus Grabhamia, eggs of Psorophora columbiae (Dyar & Knab, 1906) were also described by this technique (^{Bosworth et al., 1983}).

The eggs length of Ps. cyanescens (734.26µm) is similar to Ps. ferox (816.8 µm), but it is longer than Ps. albigenu (586.4 µm) and Ps. albipes (538.67 µm). However, the egg of Ps. cyanescens is wider compared to the mentioned species (Table 1). Hexagonal-shaped chorionic cells are similar to the eggs of Ps. cyanescens and Ps. albigenu (^{Pacheco et al., 2012}). Psorophora ferox combines hexagonal and pentagonal shapes (^{de Mello et al., 2018}), and Ps. albipes has pentagonal shapes (^{de Mello et al., 2017}). Conical tubercles are common structures in exochorion ornamentation in Psorophora (^{Pacheco et al., 2012}; ^{de Mello et al., 2017}; ²⁰¹⁸). However, tubercles vary intra and inter-species. de ^{Mello and collaborators (2018)} observed differences in tubercles number, length, and width in Ps. ferox populations from the United States, Trinidad and Tobago, and Brazil. In the present study, the long and conical tubercles of Ps. cyanescens were not measured. Further studies should consider the morphometry of mosquito egg tubercles.

The time between blood feeding and egg laying was 2.5 days for Ps. cyanescens, it is a very short period compared to seven days for Psorophora howardii (Coquillett, 1901), Psorophora ciliata (Fabricius, 1794), and Ps. ferox (^{Zhu et al., 2014}). The temperature difference could explain part of the explanation. In the present study, egg maturation was conducted in a slightly warmer environment (27 °C) compared to the previous study (26 °C). The fecundity of Ps. cyanescens and Ps. ciliata is similar, both lay an average 82 and 81 eggs, respectively (^{Zhu et al., 2014}). In contrast, Ps. howardii lays an average of 56 eggs. It was observed that a single blood feeding is sufficient for Ps. cyanescens to develop and mature eggs. In contrast, at least two blood meals are necessary for Ps. howardii oviposition (^{Gerberg et al., 1994}).

The results indicate that with SEM it is possible to identify distinctive eggs characteristics between species of the subgenus Janthinosoma. There are currently five species of the genus Psorophora, to which SEM describes eggs morphology: Ps. albigenu, Ps. albipes, Ps. columbiae, Ps. cyanescens, and Ps. ferox.