Comparative larval morphology of four Pteroptyx (Coleoptera, Lampyridae, Luciolinae) species in Thailand

Rearing

Adults and larvae of Pteroptyx spp. were collected from various riparian locations within mangrove areas in Thailand from 2023 to 2024. The research was approved for animal care and use for scientific research at Kasetsart University (ACKU67-AGR-022), and the corresponding author obtained an animal use license (U1022522558). Adults were collected from foliage using a sweep net and larvae were hand collected (Table 1). All larvae were found within the areas that contained the adult display trees. Adults were placed in circular transparent plastic boxes (four cm in diameter, six cm in height, with a one cm diameter aeration hole on the lid). A 30% honey solution on moist cotton served as food and moist cotton served as the oviposition and larval substrate. After females laid eggs on the moist cotton, the cotton with eggs was transferred to new containers to hatch at temperatures ranging from 26 to 30 °C under natural photoperiod conditions. During the egg stage, those that were unviable and started to rot were removed from the rearing containers. Assiminea (Gastropoda: Assimineidae) and Ellobium (Gastropoda: Ellobiidae) snails from the same habitat were provided as food throughout all larval stages. Contact with the fragile first instar larvae were avoided. The old cotton and consumed snails were removed every two days to prevent the growth of fungi, etc. The species identity of larvae was confirmed by both observing the development of collected larvae into adults, as adults can be identified, and observing the eggs laid by collected adults hatching into larvae. The adults were identified using the identification keys of Ballantyne et al. (2015) and Jusoh et al. (2018).

Morphological examination

The first instar larvae of all species were examined, while fifth instar were used for Pteroptyx asymmetria and P. malaccae and sixth instar for P. tener and P. valida. The use of different late instar larvae was due to the availability of rearing stock for each firefly species. Based on observations, the fourth to sixth larval instars of Pteroptyx share similar characteristics. Twelve larvae of each species were used for morphological study. To accurately document larval morphology, including soft structures, live larvae were promptly immersed in 70% ethanol before photography. The instars of the larvae were confirmed by molting during rearing to study their life cycle. Photography was conducted using a Leica DFC 295 camera attached to a Leica S8 APO Stereozoomtrinocular microscope (Leica, Heerburgg, Switzerland). Image stacks were combined into a single image, and measurements of the most representative specimen in our study series were performed using Leica Application Suite V4.2. The resulting images were then edited using Photoshop CS8 (Version 6.1) (Adobe Inc.). Described specimens were preserved and deposited at Kasetsart University (KU), Thailand.

Three larvae of each species (a total of 12 individuals) were dissected to separate and examine their mouthparts. The live specimens were placed on ice for 10 to 15 min prior to preservation in 70% ethanol. This chilling process allowed the neck membrane to relax, and become more extendible, thereby projecting the head outside and forward of the pronotum. Antennae were separated from the head, and the remaining parts were divided into two sections: mandibles and the labium-maxillae complex. The hind legs were then detached from the body. All dissected pieces were slide-mounted on glass microscope slides using modified Berlese’s medium (Amrine & Manson, 1996) before being photographed with a Leica DM 750 camera attached to a Leica ICC50W (Leica, Heerbrugg, Switzerland). Once slide-mounted, the sensory cone and the third antennal segment on the antennae and the setae on the metafemur were measured.

Numbers of retractable filaments on the holdfast organ were videotaped, photographed, and counted while placing the live larvae in water depths ranging from three to five mm using a Leica EZ4 W stereo microscope (Leica, Wetzlar, Germany).

Morphological measurements

The measurements of larval morphological characters were carried out on live specimens in order to facilitate further biological studies; therefore, some errors in the measurements of body length and body width may have occurred. However, the specimens were chilled on ice for 5–10 min to reduce activity and relax muscle tension. The larvae subjected to the ice treatment displayed a more pronounced horizontal alignment of prothorax compared to those that did not undergo treatment (Fig. S1), resulting in more accurate measurements.

Measurements of body length, body width, protergum length (PL), width (PW), and distance between each tubercle (t1–t5) (Fig. S2) were conducted on 41 larvae of four Pteroptyx species (10, 11, 9 and 11 larvae for PA, PM, PT, and PV, respectively) using a Leica EZ4 W stereo microscope (Leica). The ratio of PL and PW was calculated for morphological comparison. The t1–t5 difference within each species was analyzed using Kruskal–Wallis H-test, followed by Mann–Whitney U-test with Bonferroni correction applied for the significance level (P < 0.05). Statistical analysis was performed in SPSS V.14 software for Windows (SPSS for Windows; SPSS Inc., Chicago, IL, USA).

Taxonomic description

The characterization of Pteroptyx larvae follows Ballantyne et al. (2011); Ballantyne et al. (2015); Ballantyne & Menayah (2002) and Jusoh et al. (2018), which primarily focused on body shape, tergite shape and pigmentation, spiracle position, antennae, mouthparts, and legs. In this study, additional characters were defined, such as dissected mouthparts, small setae on the claw, prosternum pigmentation, and the number of retractable filaments in the holdfast organ. The larval characters of each species are based on the description for P. valida larvae by Ballantyne & Menayah (2002). These larval characteristics were also compared with those of aquatic species (Ballantyne et al., 2022), which described the anterior and posterior ventral areas of the meso- and metathorax of larvae as basisternum and sternellum, respectively. Supplementary redescriptions of P. valida are included to facilitate clear comparisons among species.

Taxonomy

Generic diagnosis. Found in riparian locations within mangrove or blackish water areas near river mouths; terrestrial form; elongate, slender, spindle-shaped, without laterally explanate tergal margins, laterotergites visible at sides, bearing the spiracles are visible from above (Ballantyne et al., 2011; Ballantyne et al., 2015; Fu, Ballantyne & Lambkin, 2012b), posterolateral corners of terga 1–8 round or produced depending on species; dorsal surface smooth without tubercles or with tubercles depending on species; mandible without inner teeth; antennal segment 3 much shorter than the other antennal segments, sensory cone adjacent to segment 3 short, its size and shape varied; without brush of setae from apex of tibiotarsus; similar to all Luciolinae larvae such as Colophotia species (Ballantyne et al., 2019; Ballantyne & Lambkin, 2009, explained in Fig. 517) and Curtos (Fu, Ballantyne & Lambkin, 2012a), but with dark pigmentation of tergites. The first stage of larvae can be distinguished from the texture of tergites with uniformly coarse granular and brown color on the ventral side in P. malaccae and P. tener.

Remarks. As a member of the subfamily Luciolinae, a group with diverse firefly species, most members within the subfamily have distinct characteristics on the dorsal surface. However, many of them have a ventral surface similar to Pteroptyx (Fu, Ballantyne & Lambkin, 2012a). Pteroptyx has a slender shape similar to Abscondita and Pygoluciola, but have a wide pale median line on dorsal plates and lacks heavily sclerotized tergal plates (Fu & Ballantyne, 2008, explained in Fig. 2; Nada, Ballantyne & Jusoh, 2021, explained in Fig. 2.); shape similar to Curtos, but have pigmented tergal plates with pale median line (Fu, Ballantyne & Lambkin, 2012a, explained in fig. 36). Pteroptyx larvae differ from those of Asymmetricata, Atyphella, and Emeia, which have a protergum that is wider than it is long and laterally explanate tergal margins.

General characteristics of Pteroptyx last instar larvae. (modified and expanded from Ballantyne et al. (2011); Ballantyne & Menayah (2002); Fu, Ballantyne & Lambkin (2012b) (explained in Figs. 55–60, 89–90); Jusoh et al. (2018).

Dorsal surface. Three thoracic and nine obvious abdominal segments, narrow ring at posterior end of abdominal segments IX interpreted as segment X as holdfast organ (pygopodia) (Ballantyne et al., 2022; Ballantyne & Menayah, 2002; Nada, Ballantyne & Jusoh, 2021), elongate, fusiform, slightly flattened dorsoventrally, membranous and very soft bodied, lacking laterally explanate tergal plate; dorsal body plates with dark pigmentation, but not sclerotized, P. valida are well darker than the others; pale median line clearly visible from thorax to abdominal tergite IX, divided dorsal plates into two areas, each area emarginated on posterior margin; elevated along posterior tergal margins; texture of tergal plates smooth or bearing tubercles on lateral and posterior edge, varied in different species; all tergites brown except abdominal tergite VIII light tan or with brown pigmentation; epipleural plates on mesothorax and laterotergites on abdominal segments I–VIII bearing spiracles, all abdominal spiracles visible along the sides of body, except mesothoracic spiracles visible from above (Figs. 1A, 3A, 5A, 7A).

Ventral surface. An elongate pleural suture runs from anterior margin of mesothorax to posterior margin of abdominal segment IX; meso and metathorax subdivided into two areas, an anterior basisternum with paired laterotergites bearing spiracles on mesothorax and a posterior subrectanular sternellum bearing the legs (Fig. 7F) (Ballantyne & Menayah, 2002; Fu, Ballantyne & Lambkin, 2012b); abdominal segments subdivided into median sternal area with laterosternites at the sides, and paired laterotergites with spiracles above that just beneath the terga; pigmented sternellum area and laterosternites (Ballantyne & Menayah, 2002), found in P. malaccae and P. valida or absent in P. asymmetria and P. tener.

Head. (Figs. 3A–3C, 3F, 4A, 7A, 7B) Prognathous, subparallel sided, smooth; subrectangular, dorsoventrally flattened, well sclerotized; attached to an elongated extensible neck membrane, which allows the head to be extended out of or retracted within prothorax; with median dorsal frontoclypeus, bounded laterally and posteriorly by frontal arms of the ecdysial line; parietals reflexed ventrally; dark brown, antennal bases pale; maxillae and labium fused forming a maxillolabial complex covering most of ventral head area; a pair of lateral stemmata at the base of antennae.

Antenna. (Figs. 2C, 4A, 4C, 6C, 8B) Three-segmented, smooth and glabrous; originating on lateral-apical edges of head capsule adjacent to the mandibular bases (Fig. 4A); basal segment short and widest, attached to membranous base, smooth and glabrous, elongate articulating membrane creates a two-layered covering around the retracted antenna; second segment as wide as the basal one but much longer, elongated, sclerotized, smooth and glabrous, except a long subapical hair, apex obliquely truncate; third segment very short compared to the other segments (Figs. 2C, 4C, 6C, 8B), four short and one apical setae, a small elongate SC, SC subequal in length with A3 or little longer depends on species, on ventral side.

Mandible. (Figs. 2A–2B, 4A–4B, 6A–6B, 8A) Symmetrical, strongly falcate, tapering to a truncate apex with an inner channel opening subapically on outer edge (Figs. 2B, 8A); no toothed retinaculum; covered by long setae, especially outer side, long seta parallel in median region of ventral and dorsal inner side (Figs. 6A–6B), no setae at apical point of mandible near channel opening of canal. Maximum curvature of mandibles in middle of mandible.

Labium. (Figs. 2D, 4D, 6D, 8C–8D) Loosely attached to maxilla, short and strongly sclerotized prementum, covered by dense fine setae. Postmentum heart shaped, with each anterior corner bearing a palpus; palpus short, 2-segmented (including palpiger) and bearing a long single spine.

Maxilla. (Figs. 2F, 4E, 6E, 8C, 8E) Long and robust, closely attached to labium. Stipes bearing dense long setae on outer side of maxilla. Galea large, 2-segmented, basal segment very long, lacking setae; distal segment short, narrowing distally and bearing a few short setae. Lacinia large, inner surface covered with setae. Palpus 4-segmented (including the palpifer), basal segment largest, subquadrate, longer than other segments, bearing a few long setae (Fig. 4E); distal segment cone-shaped, narrowing to a distal point, without setae, lacking a globular sensorium-type structure.

Thorax. (Figs. 1A–1C, 3A–3C, 3F–3G, 5A–5C, 5E–1F, 7A–7C, 7F–7G) Protergite subrectangular, longer than wide except P. tener, wider at the base (Fig. 5B), containing retracted head within it (Figs. 1B, 3G); meso- and metathorax subrectangular, shorter than prothorax; all thoracic tergites subdivided by median line, median line expands gradually from protergite to metatergite; each tergite elevated at posterior margin making slight slope upward (Fig. 1C). Texture of tergites differed across species, smooth without tubercles found only in P. valida; the other species have rough tergites with tubercles (Fig. S4). Color patterns of ventral surface differentiated across species (Fig. S4), no pigmented area in P. asymmetria and P. tener and with brown pigmented area in P. malaccae and P. valida; variation of pigmented areas appeared on prosternum across species; bearing well developed spiracles on mesothorax.

Legs. (Figs. 9A–9D) Four-segmented, coxae long and cylindrical; trochanters elongate; femur with a single short seta in middle, with the length of the seta varying among species; tibiotarsus as long as femora, covered with short, strong setae, with a single apical claw, tiny setae found in P. malaccae; prothoracic legs shorter than meso and metathoracic legs; legs pale with dark coxa.

Abdomen. (Figs. 1A, 1D–1F, 3A, 3D–3G, 5A, 5D–5F, 7A, 7D–7G) Segments wider than long, tapering towards end; each tergite subrectangular, tergites I–VIII divided by a median line, which divides tergites into two plates; posterior margin of each plate of tergite emarginate; surface of tergites may be smooth or rough with tubercles on posterior margin of tergite plates depend on species; pleural areas well developed, abdominal segments I–VIII subdivided; pleural areas of segments IX and X reduced; color pattern similar to that of thorax; brown abdominal tergites I–IX except VIII appearing different color patterns in different species, bearing a pair of light organs on ventral side of segment VIII; segment IX elongate slender; segment X a narrow ring carrying holdfast organ, numbers of retractable filaments of holdfast organ differed in different species, P. valida has more than 10 filaments (Figs. 10A–10D), bearing different outline on the base of holdfast organ, a small spine underneath the holdfast filament (Fig. S3).

Diagnosis. Pteroptyx asymmetria can be distinguished from other Pteroptyx species, except P. tener by its smaller size; differing from P. valida by bearing tubercles on tergite 1–11 margin, with higher tubercles than those of P. maipo, having much longer protergum length than width; anterior margin of protergum angles not round as in P. valida and P. maipo; ventral surface dull color with no dark pigmentation; prosternum darkly pigmented except longitudinal median region (Figs. 1B, 1E), similar to P. malaccae and some P. tener (Type I, Fig. S4); abdominal tergite VIII light tan on lateral margin where located a pair of light organs, very similar to P. malaccae; holdfast organ bearing 10 retractable filaments, differing from P. valida.

Description, fifth instar larvae. (Figs. 1–2) 6.71–8.41 mm long; 1.31–1.69 mm wide; long protergum length than width (0.77–1.33 mm long; 0.73–0.99 mm wide; length:width 1.05–1.48); bearing tubercles on tergite 1–11 margin (Figs. 1A, 1C), six small tubercles (white arrows) on anterior margin and six large tubercles (black arrows) on posterior margin of protergites, six large tubercles on lateral and posterior margin of meso- and metatergites, four big tubercles on posterior margin of abdominal tergites; tubercles well developed around protergite plates making anterior shaped angles of protergum, anterior margin of protergum shaped angles; dark marked prosternum except longitudinal median region, sometimes (20%) found a pigmented diamond shape in posterior region; femora have one single short setae in middle, 0.26 mm long, longer than a half of tibiotarsus (Fig. 9A); abdominal tergites I–IX brown, except tergite VIII light tan on lateral margin where located a pair of light organs; no color pigmentation on ventral surface except slightly pigmented on coxa; holdfast organ bearing 10 retractable filaments, with two curved outlines on the base sides; SC subequal in length with A3 (Fig. 2C).

Description, first instar larvae. (Figs. 1G, 1H) 0.95–1.08 mm long; all dorsal plates uniformly coarse granular except tubercle areas, tubercles on all tergites formed except small tubercles on protergite, each tergites elevated at the posterior margin; median line on thoracic segments and abdominal tergites I–VII; ventral surface light brown except brown pigmentation on prosternum; no color pigmentation on ventral surface of meso- and metathorax; uniformly fine granular distributed on ventral area.

Diagnosis. One of three species of Pteroptyx known from Thailand and P. maipo with tubercles on tergite 1–11 margin, differing from both P. valida; protergum longer than wide, considerably similar to P. tener and P. maipo, but with protergum length not as long as P. asymmetria; having protergum shaped angles on anterior margin and distinguished from P. valida and P. maipo which have rounded shaped angles; one of three species possessing brown pigmented areas on the ventral side, similar to P. maipo and P. valida, differing from P. valida by darker pigmentation; prosternum dark pigmented except longitudinal median region, distinguished from some P. asymmetria (20%) and P. tener (Type I) by bearing a pigmented diamond shape in posterior region; claw bearing very small setae that differed from the other species; abdominal tergite 8 with brown; holdfast organ bearing 10 retractable filaments, differing from P. valida.

Description, fifth instar larvae. (Figs. 3–4) 8.27–10.97 mm long; 1.78–2.03 mm wide; protergum length longer than width (1.25–1.58 mm long; 1.05–1.23 mm wide; length:width 1.03–1.23); short tubercles on tergite 1–11 margins (Figs. 3A–3B), twelve tubercles around protergite, six tubercles on lateral and posterior margins of meso- and metatergites, coxa brown pigmented on ventral surface; femora bearing one single short setae in middle, 0.43 mm long, longer than three-quarters of tibiotarsus (Fig. 9B), claw bearing minute setae; four tubercles on posterior margin of abdominal tergites; abdominal tergite VIII pale with brown pigmentation (Figs. 3A, 3D); abdominal tergites I–IX brown except tergite VIII tan colored on lateral margins adjacent to the paired larval light organs, also visible in ventral view; holdfast organ bearing 10 retractable filaments; SC = 2/3A3 (Fig. 4C).

Description, first instar larvae. (Figs. 3H–3I) 2.25 mm long; all dorsal plates uniformly coarse granular except tubercle areas, tubercles on all tergites formed incompletely; median line on thoracic segments and abdominal tergites I–VII; ventral side with dark pigmentation on prosternum; uniformly fine granular distributed on ventral area.

Diagnosis. One of three Pteroptyx with tubercles on the margin of tergites 1–11; distinguished from P. asymmetria and P. malaccae by subequal protergite; a light tan ventral surface distinguishes this species from P. maipo, P. malaccae and P. valida by lacking brown pigmented areas as well as a brown pigmented area in the medial region of abdominal tergite VIII; holdfast organ bearing ten retractable filaments.

Description, sixth instar larvae. (Figs. 5–6) 5.78–8.41 mm long; 1.24–1.82 mm wide; length:width 0.99–1.20); small size (7.35–8.34 mm long; 1.52–1.84 mm wide); bearing tubercles on margins of tergites 1–11 (Fig. 5A–5B), six small tubercles on anterior margin and six large tubercles on posterior margin of protergite, six large tubercles on lateral and posterior margin of meso- and metatergites, four large tubercles on posterior margin of abdominal tergites; anterior margin of protergum shaped angles; pale abdominal sternites lacking dark pigmentation (Fig. 5E). Ventral color pattern of prosternum variable: Type I (Fig. 5C, Fig. S4) prosternum darkly pigmented except longitudinal median region (four of six larvae), Type II (Fig. 5E, Fig. S4) prosternum ventral surface uniformly brown except a deeply emarginate pale area region at the anterior margin (two of six larvae); ventral surface from mesothorax rearward, without pigmentation; femora have one single short setae in middle, 0.20 mm long, equal half of tibia (Fig. 9C); abdominal tergites I–VII and IX brown, tergite VIII light tan in color, with pigmentation restricted to only anterior and posterior margins (Fig. 5D); ventral surface without pigmentation; holdfast organ bearing ten retractable filaments (Fig. 10C); SC = 3/4 A3 (Fig. 6B).

Description first instar larvae. (Figs. 5G–5H). 0.95–1.88 mm long; dorsal and ventral brown; all dorsal sclerites uniformly coarse granular, tubercles absent; median line on thoracic segments and abdominal tergites I−VII; ventral side very pale except brown prosternum; ventral surface uniformly finely granular.

The larval characters of the first to third, as well as last instar larvae were examined and comprehensively described following the previous work of Ballantyne & Menayah (2002). A supplementary redescription is provided here for comparison to the other Pteroptyx species known from Thailand in the above sections.

Diagnosis. One of two Pteroptyx species that have smooth tergites, lacking tubercles on all tergites, thereby differing from P. asymmetria, P. malaccae and P. tener; very similar in dorsal and ventral shape and coloration to P. maipo but differing in having a pigmented diamond pattern on the posterior region of the prosternum, pale abdominal tergites VIII and pale lateral margins of abdominal tergite IX; ventral surface darkly pigmented; holdfast organ bearing more than 10 retractable filaments and with dark outline on the base sides.

Redescription, sixth instar larvae. (Figs. 7–8) 9.67–12.28 mm long; 1.83–2.54 mm wide; protergum length greater than width (1.2–1.80 mm long; 1.13–1.53 mm wide; length:width 1.07–1.18); tergites smooth, thoracic and abdominal sternites darkly pigmented, prosternum bearing a small pigmented diamond shape in the medial posterior region (Figs. 7B, 7F); anterior margin of protergum bluntly rounded (Fig. 7A); bearing dark pigmentation on ventral side on coxa and medial abdominal sternite; femora have one single short setae in middle, 0.39 mm long, longer than a half of tibia (Fig. 9D); dark brown abdominal tergites I–IX except tergite VIII which is light tan in the lateral regions at location of larval light organs (Fig. 7D); holdfast organ bearing more than 10 retractable filaments (Fig. 10D, Fig. S3A) and with dark outline at the base (Figs. 7D, 7F, 10D), with four curved outlines on the base sides; SC subequal in length with A3 (Fig. 8B).

Redescription, first instar larvae. (Figs. 7H–7I). 1.48–2.54 mm long; all dorsal sclerites uniformly coarse granular, tergites lacking tubercles; median line on thoracic segments and abdominal tergites I–VII; bearing dark pigmentation on ventral surface of prosternum, same as the sixth instar.

A key to species of Pteroptyx last instar larvae known to occur in Thailand

Morphological comparison among Pteroptyx species

Several differences in the morphological characters of Pteroptyx larvae were investigated (Table 2, Fig. S4). The characters determined to be important for species identification are protergum shape, tergite texture and coloration. Variation in prosternum coloration was found in the rough tergite species. The scatter plot (Fig. 11A) of protergum length and width contrast with the variation in size among Pteroptyx species. Pteroptyx valida larvae have the largest protergum, followed P. malaccae, P. asymmetria and P. tener. Pteroptyx asymmetria exhibits the largest protergum length and largest PL:PW ratio, while P. tener is the smallest (Fig. 11B).

As three of the four species bear tubercles on all margins of the protergum, P. asymmetria, P. malaccae, and P. tener were analyzed for the distances between the tubercles in each species. T1 to T5 in each species were significantly different (Figs. 11C–11E; Kruskal–Wallis H-test: χ2 = 26.120, df = 4, P = 0.000 for P. asymmetria; χ2 = 24.537, df = 4, P = 0.000 for P. malaccae; χ2 = 31.754, df = 4, P = 0.000 for P. tener). The distances of t1, t2, t3, t4 and t5 of P. asymmetria and P. malaccae follow a similar trend with t2 being the shortest and t5 being the longest, in contrast to P. tener.