﻿Life cycle and description of the immature stages of a terrestrial firefly endemic to Mexico: Photinusextensus Gorham (Coleoptera, Lampyridae)

﻿Abstract The life cycle, morphology, and bionomy of Photinusextensus Gorham, 1881, an endemic species of Mexico, are described. Redescriptions of adults (male and female) are also presented. Larvae were reared to the adult stage from eggs laid by females collected at the El Pedregal de San Ángel Ecological Reserve, south of Mexico City. The activity period of adults of P.extensus begins at the end of July and finishes by the end of August. Females lay between 3 and 198 eggs. Larvae hatch from the eggs after a period of 11 to 71 days, undergo 6 larval instars and a pupal stage in an annual cycle. Morphological characters of the sixth larval instar of P.extensus are compared with those of several other genera assigned to the tribe Photinini. Knowledge of the natural history of firefly larvae is relevant since most species do not feed as adults and therefore depend on resources acquired during the larval stage.


Introduction
Fireflies belong to the family Lampyridae Rafinesque, 1815, and show a wide-ranging phenotypic and ecological diversity (Riley et al. 2021). Currently, there are more than 2400 described species with a worldwide distribution (Martin et al. 2019;Ferreira et al. 2020;Zaragoza-Caballero et al. 2020;Riley et al. 2021;Silveira et al. 2022). The highest species diversity is found in the Neotropical region (Costa 2000). Fireflies include nonluminous and luminous adults, luminous larvae and the females of some taxa are flightless (Branham 2010;Lewis et al. 2020). They inhabit wetlands, grasslands, forests, agricultural fields and urban parks (Lewis et al. 2020). Many fireflies are strongly associated with particular habitats and vegetation types (Faust 2017).
Firefly species can be either diurnally or nocturnally active. Diurnal species generally do not have light organs as adults and rely on pheromonal and visual cues (Ohba 2004;Branham 2010). Luminous species are nocturnal or crepuscular, with bioluminescent signals produced from photic organs of various shapes and sizes located on abdominal ventrites. These visual signals are typically used in sexual signaling to communicate species identity and facilitate pair formation (Branham 2010).
Firefly larvae can be aquatic, semiaquatic or terrestrial and can be found along the margins of streams and ponds as well as in leaf litter or rotten logs (Branham 2010). All known larvae are luminous and emit glows of varying duration. They are predatory on snails, earthworms and other soft-bodied prey (Lewis et al. 2020). The adults of most species do not feed and therefore rely on resources gathered during the larval stages (Lloyd 2002;Vaz et al. 2020). Multiple genera endemic to the Neotropical region have no larval or pupal descriptions. The few species that have been studied have different life histories (Vaz et al. 2020).
Photinus Laporte, 1833 is the most diverse genus of the subfamily Lampyrinae with more than 300 described species (McDermott 1964;Zaragoza-Caballero et al. 2020). Members of this genus live in a variety of habitats (from tropical dry forests to temperate and tropical montane cloud forests). Species range from the United States to Argentina. One species, recently collected in Spain, was described as new (Zaragoza-Caballero and Viñolas 2018) but then synonymized with the South American species Photinus signaticollis (Blanchard, 1846) (Koken et al. 2022).
In the past 20 years the number of known Photinus species has increased due to the description of new species from Mexico (Zaragoza-Caballero 2000, 2007, 2015Zaragoza-Caballero et al. 2020). This fact contrasts with the lack of knowledge of the larval stages and the natural history of these organisms. Until now, no study has accurately documented the life cycle duration of a Photinus species. This lack of knowledge is the result of the difficulty of collecting mated females, as well as challenges associated with rearing larvae under laboratory conditions. Moreover, some species are known to spend several years in the larval stage, and the time fireflies need to complete their life cycle depends on the geographical region and the availability of larval food (Buschman 2017).
As for most genera in the family Lampyridae, Photinus larvae are poorly studied. Buschman (2017) observed that the first instars of this genus are smaller compared to those of Photuris Dejean, 1833, but no descriptions are provided. Most research documenting the natural history of Photinus focuses on the adult stage. These studies include courtship in males (flash communication) (Wing 1991;Viviani 2001;Faust and Weston 2009;Faust 2010), nuptial gifts (Lewis et al. 2004a(Lewis et al. , 2004b, the utilization of ejaculate-derived proteins to nourish developing oocytes in certain species (Demary 2005) and the size of signal detection and emission organs .
This paper documents the life cycle and larval morphology of a Photinus species for the first time. Photinus extensus Gorham, 1881 is an endemic species of Mexico. Its known distribution includes the state of Chiapas, Hidalgo, Mexico, Morelos and Mexico City (Zaragoza-Caballero et al. 2020). A reduced population of this firefly was also found in El Pedregal de San Ángel Ecological Reserve, South of Mexico City. The life cycle of P. extensus is herein presented, with descriptions of larval instar 6. Some characteristics of instars 1 and 3 are mentioned; the adult was described as well.

Collection
Adults of P. extensus were collected in the buffer zone of the El Pedregal de San Ángel Ecological Reserve (19°19'28.82"N,99°11'20.95"W); this zone is between the core zone and the urban area of Mexico City, it is totally in the territory of the Universidad Nacional Autónoma de México. El Pedregal de San Ángel Ecological Reserve is located at the Southeast of Mexico City in the central Campus of the (UNAM) (Fig. 1). This community developed on a basaltic lava substrate approximately 1,670 years ago (Lot and Camarena 2009); the type of vegetation corresponds to a xerophytic shrub (Rzedowski 1978). The climate is sub-humid tempered with summer rains and an annual average precipitation of 833 mm; the annual average temperature is 15.5 °C (Orozco-Segovia et al. 2009). Fourteen adult females and 70 adult males of P. extensus were collected on August the 3 rd , 8 th , and 10 th of 2018, between 19:30 and 21 h. Adult specimens were located by their bioluminescence in the undergrowth, where the dominant plant species is Pittocaulon praecox (Cav.) H. Rob. & Brettel (Asterales: Asteraceae). Other collections were made at the same site between June and July 2019, and five larvae were obtained. These developed into two female pupae and three males. Larvae, pupae, and adults were fixed in 70% ethanol for their preservation.

Rearing in the laboratory
To observe the reproductive activity of P. extensus, adult specimens collected were divided into 14 groups consisting of five males and one female were placed in an 8 × 15 cm plastic container; peat moss substrate was added to simulate their natural environment. After oviposition, eggs were placed over a gauze patch in a 50 × 100 mm Petri dish, and moistened every 48 hours with an antimycotic solution based on Nistatine diluted in water (1/10). After eclosion, larvae werepartitioned into groups of five in separate Petri dishes (5×10 mm). To avoid dehydration, a filter paper layer was added and moistened every two days. This filter paper was replaced every week. Starting with the fourth larval instar, each larva was placed in a separate Petri dish (5×10 mm), with half of the dish covered with the filter paper, and the other half filled with sterilized dry sawdust. Following previous studies (Archangelsky and Branham 2001;Archangelsky 2010), larvae were fed small pieces of earthworm Eisenia fetida (Savigny, 1826) (Haplotaxida: Lumbricidae). Dead or partially consumed prey were removed from the Petri dishes every two days to keep containers clean. Pupae were maintained in Petri dishes (5×10 mm) at room temperature with sterilized dry sawdust until they completed their development. Specimens representing the different larval instars, pupae, and adults, were preserved in 70% ethanol for reference and subsequent study.

Morphological study
Adults were identified using original descriptions, literature (Zaragoza-Caballero et al. 2000), and by comparison with photographs of type specimens (Natural History Museum of London, BMNH) ( Fig. 2A-F) and specimens identified by experts deposited at the Colección Nacional de Insectos (CNIN), Instituto de Biología, UNAM, terminology of internal genitalia of females followed Silveira et al. (2022). Juvenile stages and adults of P. extensus were examined under a Zeiss stereoscopic microscope (Discovery V8) with a 1× objective lens coupled with 16× eyepieces. Larval heads of each instar were separated from the body and immersed in 10% KOH solution, the mouthparts were dissected under a stereoscopic microscope and placed in glycerin on slides for observation. Description of the distribution of the setae was made for the last instar larva following Branham and Archangelsky (2000) and Archangelsky (2010). Pygopodial structure for the last instar larva was interpreted using Fu et al. (2012). Redescription of the adult of P. extensus was made based on collected material. For the morphological description of larval instars, we followed the terminology of Novák (2018a) and Fu et al. (2012). A table 1 was made to compare morphological larval characters of Photinini (Pyractonema, Pyropyga, Lucidota, Lucidina, Phosphaenus and Photinus) (Branham and Archangelsky 2000;Archangelsky and Branham 2001;Archangelsky 2010;Novák 2018b), after Archangelsky (2010) and Kawashima (2017).
Images were taken with an AxiocamMRC5 camera attached to a Zeiss Axio Zoom V16 microscope with an objective lens Plan NeoFluar Z, 1×10.25 FWD 56 at the Laboratorio de Microscopía y Fotografía de la Biodiversidad II, Instituto de Biología, UNAM. Larvae were examined and imaged with a Hitachi SU1015 scanning electron microscope at the Laboratorio de Microscopía y Fotografía de la Biodiversidad I, Instituto de Biología, UNAM.

Description of pre-imaginal stages.
Egg (Fig. 5A, B). Semispherical shape, whitish, with a diameter of approximately 290-300 µm (Fig. 5A). Surface with concavities that differ in size and shape (4-10 µm), evenly distributed, some present in aggregations (Fig. 5B). As time elapsed, the surface of the eggs became more transparent, allowing the observation of the larvae before hatching.  (Figs 6A, B, 7A-H, 8A-G, 9A-F). Description. Elongate, tapering body, dorso-ventrally flattened, length 12.27-18.18 mm; integument of granular appearance; tergites from protergum to abdominal segment IX divided by sagittal line in dorsal view. Tergites with two lateral pale stripes that run throughout the body to the VIII segment, more sclerotized than the sternites, with clearly visible setae on the posterior margin of tergites VII to X; the last tergum completely dark except the lateral margins paler; anterior margin of the first head segment with two fossae (sensorial or glandular) paler and bigger than the rest of the punctations of the segment. Membranous pleura except for a dark sclerotized area around the spiracles, without apparent setae. The ventral surface is flexible due to the intersegmental membranes. Mesothoracic and abdominal pleural areas of segments I-VIII with bilabiate spiracles. Head capsule. Prognathous; slightly visible when retracted into prothorax due to the transparency of the protergum; extensible neck membrane covered in extremely short spines forms a two-layer envelope around the head; partially retractable within the prothorax; completely sclerotized, small, wider (0.88-1.54 mm; 1.2± 0.27 mm) than long (0.68-1.09 mm; 0.92 ± 0.17 mm), flat, sides almost parallel; stemmata on each side, with an almost transparent spot located posteriorly to the stemmata; clypeus and labrum fused forming the clypeo-labrum covering base of the mandibles in dorsal view; maxillae and labium connate forming maxillolabial complex covering most of the ventral cephalic area; epicranial suture dark, U-shaped, with a very short epicranial stem, frontal arms Vshaped (Figs 7A, 8A). Epipharynx formed by two oval plates, without setae, that project centrally beyond the anterior margin of the head. Hypopharynx with short setation.

Sixth instar larva
Antenna. Trimerous, located on the distal margin of the epicranial plate; partially retractable into the antennal socket; three-segmented, basal antennomere and second antennomere (0.42-0.55 mm; 0.48 ± 0.05 mm) elongated, and a third segment (the flagellum) short (0.24-0.31 mm; 0.26 ± 0.03 mm); adjacent sensorial cone present; basal antennomere with two long setae in the anterior mid, almost entirely covered by moderately dense, second antennomere with long setae close to apex and entirely covered by dense smaller finer setae, third antennomere with long setae from base to apex, with short setae on the anterior margin (Fig. 7B, 8B).

Maxilla.
Consisting of five parts, attached to lateral margins labium forming a maxillo-labial complex. Cardo elongate, irregular shape, with four setae in ventral surface, on long setae in posterior margin. Stipes elongated, ventrally covered with erect setae, with three long stout setae placed radially on the ventral apical region. Galea present, with two segments, the first longer and stouter than the apical, which is triangular (Fig. 7C, D). Lacinia covered with brush of long setae on outer lateral margin. Maxillae with three-segmented palpi, basal segment long (0.57-0.71 mm; 0.66 ± 0.06 mm) covered by setae in mid-region, segment II wider (0.30-0.40 mm; 0.35 ± 0.04 mm) than long (0.12-0.16 mm; 0.14 ± 0.01 mm); apical segment cylindrical (0.10-0.14 mm; 0.12 ± 0.01 mm) with numerous setae from base to mid region; (Figs 7E, F, 8C, D). Labium. Closely attached to maxilla, formed by prementum, mentum and postmentum. Prementum heart-shaped, surface covered with numerous short setae and two long setae close apex; labial palpi with two segments, basal palp subquadrate with few setae in mid-region, distal palp conical without setae; mentum with one pair of setae on anterior third and one pair of setae on posterior third; postmentum elongate, slightly sclerotized at the medial base, laterally united by membranes to the cardines; with a setae on each side near the base.
Mandible. Symmetrical, falcate, strongly sclerotized, with an internal channel opening subapically on outer edge. Penicillus well-developed. Retinaculum short and rounded, present only as a blunt protuberance on basal third of the mandible. Densely covered by fine setae on the external margins basely, basal half on inner margin of mandible covered with a brush of stout setae, being longest on the retinaculous protuberance (8E, F); mesal margin serrate.
Thorax. Protergum wider (2.43-3.81 mm; 3.14 ± 0.57 mm) than long (1.54-2.59 mm; 2.11 ± 0.43 mm), subsemicircular, wider posteriorly, rounded at posterolateral corners, covering the retracted head. Meso-and metatergum subrectangular three times wider than long, delimited by a pleural suture elongate barely evident from the laterotergites. Lateral areas of meso and metathorax scarcely sclerotized, composed of two laterotergites, the anterior with a well-developed spiracle on the mesothorax. Episterna extending from the anterior part to the lateral part of the coxae; epimeron forming a little sclerotized stripe, parallel to the coxae.
Legs. Pentamerous, the first pair of forelegs slightly shorter than the second and third. Coxae short (0.76-1.16 mm; 0.98 ± 0.18 mm), cylindric, widely separated at the base, decumbent; coxal-trochanteric membrane reaching about 1/3 of the coxal length. Trochanters pentagonal, joining the femur obliquely (0.51-0.82 mm; 0.71 ± 0.13 mm). Femur narrow and cylindrical in lateral view. Tibiotarsus narrowing distally with stout setae. All legs with a double row of long setae in the inner margin, numerous short setae in the outer margin: pretarsus claw-like with two setae at base (Figs 7G, H, 8G).
Head. Totally covered by the pronotum in dorsal view. Large eyes, located at the sides of the head; antennae in front of the eyes, nearer the frontal center, mouthparts visible in ventral view.
Thorax. Pronotum wider than long, semicircular, totally covering the head. Meso and metanotum shorter, subrectangular, bearing the elytra sidewards. All pairs of legs free, visible in ventral view. Spiracles present in the pleural areas of mesothorax.
Abdomen. Abdominal segments subrectangular, wider than long, spiracles present on abdominal pleural areas of segments I-VIII. Light organ on sternites V-VI.
Life cycle (Fig. 11). In their natural habitat, adults of P. extensus are active from early July, when the first males can be observed. Bioluminescent activity begins at dusk, at approximately 20:00 h, and diminishes considerably an hour later. Male flight does not exceed 2 m in height. Females are brachypterous and perch in the undergrowth approximately 50 cm from the ground. Males flash every 4.5 seconds, flying in an arc when illuminated. When males detect a female, they wait for an intense flash as a response, which is brief. The flash intervals are of 10 to 20 seconds. Males react by flying lower and towards the female. Groups of 3 to 5 males commonly compete with each other to get the female first, to mate with her. Two types of competition were observed among males: 1) a mating ball: four or more males cover the copulating pair and try to dislodge the copulating male to gain access to the female, and 2) males using their pronotum as a lever to pry a copulating male from the female. In the laboratory, copulation was observed to last from between 2 to more than 4 hours.
During oviposition, females bend their abdomen and place the apical part of it on the substrate. Eggs are laid superficially or buried, randomly distributed, individually, or in groups (up to aggregates of 50). The number of eggs deposited by each female varied from three to 198. Eggs emit a faint bioluminescence since they are oviposited, which is only perceptible to the human eye in complete darkness. In total, 956 eggs from 13 females were obtained.
Under lab condition P. extensus completed its development in approximately 12 months, from oviposition to imago. The egg stage under laboratory conditions had a duration of 11 to 70 days, with mortality of n = 144 eggs (15%).
Photinus extensus undergoes six similar larval instars that differ in both size and color (Fig. 4). Cannibalism among larvae during rearing was not observed. The only food larvae consumed was the earthworms provided. There was no synchronization among larvae during the progression of larval instars, which started at the end of August until the beginning of July. In captive conditions, the process of ecdysis from one stage to the other varied among individuals. The first larval instar had a duration of 14 to 153 days, where mortality was 60% (n = 491) among the eggs that hatched. The second larval instar had a duration of 14 to 172 days, with mortality of 61% (n = 199). The third larval instar had a duration of 15 to 140 days; mortality was 71% (n = 87). The fourth larval instar had a duration of 17 to 140 days, with a mortality of 52% (n = 20). The fifth larval instar had a duration of 24 to 192 days (n = 18) and the last larval instar had a duration of 53 days (n = 8). Pupation had a duration of 7 days in July, and according to the observations in the field, pupae were found under pyroclastic rocks.

Discussion
Photinus extensus has six instars; they are very similar and only differ in size, color and the degree of the body sclerotized and presence of setae. Instar III differs from I and VI by a trapezoidal pronotum (Fig. 5) and an exposed head. The periods of time of the instar I-V are variable in different specimens, ranging from 14 and 192 days; the last instar period in different specimens is constant, approximately 50 days; the pupa is completely developed in seven days.
Frequently, the identification and description of larvae in the tribe Photinini is based on the characters present in the final larval instar, mostly body shape, color pattern, head capsule features, and the morphology of mouthparts (Archangelsky 2010). There are both similarities and differences between larvae belonging to the tribe Photinini, including P. extensus (see Table 1). The larval character suite found in P. extensus is most similar to those found in Pyractonema, Lucidota, and Pyropyga. The major exception to this is the number of segments in the maxillary palp. Although this last character is shared with the genus Phosphaneus, it differs in the opening of the mandible channel. P. extensus differs from Lucidina in the number of segments in the maxillary palp, and the number of retinacula of the mandible. There are some patterns among the larvae described in Photinini; the shape of the body is narrow and parallel and the pronotal shape is semicircular or semioval and the opening of the mandible channel is at the inner margin subapical.
The distribution of the setae is different between genera (Table 2), chaetotaxy will be useful in Lampyridae as a tool to distinguish between immatures, also as a source of informative phylogenetics characters (Ballantyne et al. 2019;Riley et al. 2021;Vaz et al. 2021). However, the few studies on the morphology of immatures in Lampyridae and the lack of knowledge of chaetotaxy hinder comparison between fireflies and makes the elaboration of more detailed hypotheses very difficult. The larval characters have shown to be important to clarify the phylogenetic relationships as Archangelsky (2010) mentioned. However, it seems to be a difficult task to get a good dataset of larval characters of Lampyridae.
The length of the pupal stage varies slightly in Photinus. The pupal stage of P. extensus and P. carolinus has a duration of six days (Faust 2010). Nevertheless, the pupa of P. carolinus is present during May, while that of P. extensus is present in July. Another difference is that the pupae of P. extensus observed in the field were found under pyroclastic rocks, in contrast with P. carolinus, which has been reported to occur under leaf-litter, near rotten logs, or moss (Faust 2010). Until now, little information has come to light about the pupae in other genera of Photinini (Archangelsky and Branham 2001). In other genera like Aspisoma Laporte, 1833, of Cratomorphini, the pupal stage is similar, occurring during a short period between six and ten days (Costa et al. 1988;Archangelsky 2004). No information.

Mandible
With patches of dense pubescence in the basal part in ventral Margin covered by dense pubescence. With patches of dense pubescence in the basal part in ventral view. Mid-region covered by a single row of long setae.
With patches of dense pubescence in ventral view. Mid-region covered by only one row of setae.
Mid-region covered by only one row of long setae. One long seta close to the apex.

Cardo
With 4 long setae in ventral surface and one long seta in the posterior margin.

Maxillary palpomeres
Basal palp covered by setae in the mid region. Distal palp with setae from base to mid region.
Basal palp covered by setae. Palp II with some setae. Last palp without setae.
Basal palp with long setae from the mid region to the apex. Palp II with shorter setae. Distal palp without setae.

Labial palpomeres
Basal palp with few setae in mid region. Distal palp without setae.
Basal palp with few long setae. Distal palp with one long seta.

Prementum
Surface with many setae. Two long setae close to apex.
Dorsal and ventral surface with many setae.
With two basal regions of very fine setae, with longer setae on the palp segment.

Submentum
With two long setae in mid region.
With two long setae in the basal middle.
No information.
Antennomeres Apex with two long setae. Antennomere III with long setae from base to apex.
All antennomeres covered by setae. Antennomere III with many short setae.
Basal antennomer with mid region covered by setae. Anterior region with longer setae. Antennomere II covered evenly by long setae. Antenomere III with short setae.

Legs
With a double row of long setae in the inner margin. Outer margin with many setae. Pretarsus with two setae at the base.
With a double row of long setae in the inner margin. Pretarsus with two setae at the base.
With a double row of long setae in the inner margin. Pretarsus with two setae at the base.

Abdomen
Sternites with two long setae in mid-region. Sternites with two long setae in mid-region.
No information.

Conclusion
The complete life cycle of Photinus extensus, including descriptions of egg, larvae, and pupa, was documented for the first time. Larvae were reared in laboratory conditions to the adult stage from eggs. The six instar of P. extensus are very similar; they differ only in size and in the sclerotized degree. Among Photinini larvae there are not many differences, differing in the number of segments in the maxillary palp and in the number of retinacula of the mandible; the shape of the body and pronotum, and the opening of the mandible channel follow a similar pattern. Life cycle information is essential to carry out protection and conservation actions for insects that are very sensitive to environmental changes, like fireflies. For example, the species that do not produce light are easily overlooked and the information about their life cycle is deficient. This results in "Data Deficient" categorization in evaluations of extinction risk. Thus, more studies are needed in which the life history, habitat associations, and microhabitat are detailed (Fallon et al. 2021). Also, it is necessary to understand that requirements of larvae are different to those of adults to have an integral vision in the actions of protection of the fireflies.