Research Article |
Corresponding author: Mariano C. Michat ( marianoide@gmail.com ) Academic editor: Kelly Miller
© 2017 Mariano C. Michat, Grey T. Gustafson, Johannes Bergsten.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Michat MC, Gustafson GT, Bergsten J (2017) Larval description and chaetotaxic analysis of Dineutus sinuosipennis Laporte, 1840, with a key for the identification of larvae of the tribe Dineutini (Coleoptera, Gyrinidae). ZooKeys 718: 95-114. https://doi.org/10.3897/zookeys.718.20726
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The larvae of the Malagasy whirligig beetle Dineutus sinuosipennis Laporte, 1840, identified using DNA sequence data, are described and illustrated for the first time, including detailed morphometric and chaetotaxic analyses of selected structures and a description of larval habitat. Larvae of the genus Dineutus Macleay, 1825 are diagnosed, and a key to identify the genera of the tribe Dineutini is presented. Larvae of Dineutus exhibit the characters traditionally recognized as autapomorphies of the Gyrinidae: body less sclerotized, egg bursters located on the parietal, one additional sensorial plate on the third antennomere, cardo and lacinia well developed, prementum completely divided, abdominal tracheal gills, and four terminal hooks on the pygopod. They also share with larvae of the other Dineutini genera these putative synapomorphies: numerous minute pore-like additional structures on the ultimate maxillary and labial palpomeres, coxal primary seta CO12 inserted submedially, and trochanteral primary seta TR2 absent. Larvae of Dineutus can be distinguished from those of other known genera of Dineutini by the posterior margin of the lacinia not dentate, tracheal gills plumose, parietal seta PA5 inserted relatively far from setae PA7–9, mandibular pores MNb and MNc inserted relatively far from each other, and tarsal seta TA1 inserted submedially.
Adephaga , whirligig beetles, larva, sensilla, DNA-based association
The genus Dineutus Macleay, 1825 comprises 92 relatively large-sized species and has a near global distribution, being absent from Europe and, notably, from South America (
Larval morphology of Dineutus is poorly known. Larvae of a vast majority of species are unknown, and the few described ones are insufficiently documented.
In recent years, several descriptions of gyrinid larvae were published, emphasizing not only general morphology but also including chaetotaxic analyses in the following genera and subgenera: Macrogyrus (Andogyrus) Ochs, 1924 (
In the present paper, a detailed description of all larval instars of the genus Dineutus is provided, including, for the first time, morphometric and chaetotaxic analyses of the cephalic capsule, head appendages and legs of D. sinuosipennis. Comparisons with other gyrinid genera for which the larvae have been described in detail are also provided, and an identification key to separate all instars of the known genera of the tribe Dineutini is presented. Despite adults being collected readily by the hundreds, larvae of many gyrinid species remain frustratingly elusive. For this reason, we also provide a description of the precise habitat where larvae of D. sinuosipennis were collected to aid future collection.
The descriptions provided in this paper are based on two specimens of instar I, three of instar II and three of instar III collected in Madagascar, at the locality described below.
There are three species of Dineutus known from Madagascar: D. proximus Aubé, 1838, D. sinuosipennis and D. subspinosus (Klug, 1834). Only adults of D. sinuosipennis were found at the locality where the larvae were collected, but for unambiguous association we extracted DNA from four larvae of instar III and sequenced mitochondrial cytochrome c oxidase subunit I (COI) using the primers ‘Jerry’ (
PCRs were set up in 25 ul reactions using Illustra Hot start mix RTG (GE Healthcare, Little Chalfont, Buckinghamshire, UK), 1 ul each of the Primers (10 uM), 2ul of DNA template and 21 ul water. Thermal cycling profile included the following steps: 95 °C for 5 min followed by 40 cycles of 95 °C for 30 s, 50 °C for 30 s and 72 °C for 60 s, and finally 72 °C for 8 min. Products were cleaned with the ExoFast method, using a combination of Exonuclease 1 and FastAP (ThermoFisher Scientific, Waltham, MA, USA). Cleaned products were sequenced using Big Dye version 3.1 on an ABI 3130XL Genetic Analyzer. The molecular lab work was carried out by the Centre for Genetic Identification (
Forward and reverse reads were combined into contigs in Sequencher (version 4) and primer regions were removed. The sequences were trimmed to the 740bp length fragment used by
The larvae were cleared in lactic acid, dissected, and mounted on glass slides in polyvinyl-lacto-glycerol. Microscopic examination at magnifications up to 1,000× and drawings were made using an Olympus CX31 (Olympus Corporation, Tokyo, Japan) compound microscope equipped with a camera lucida. Drawings were scanned and digitally inked using a Genius PenSketch tablet (KYE Corporation, Taipei, Taiwan). The material is held in the collection of the senior author (Laboratory of Entomology, Buenos Aires University, Argentina).
We employed the terms used in previous papers dealing with the larval morphology of Gyrinidae (
Primary setae and pores were distinguished in the cephalic capsule, head appendages and legs. Sensilla were coded by two capital letters, in most cases corresponding to the first two letters of the name of the structure on which they are located, and a number (setae) or a lower case letter (pores). The following abbreviations were used: AN, antenna; CO, coxa; FE, femur; FR, frontoclypeus; LA, labium; MN, mandible; MX, maxilla; PA, parietal; PT, pretarsus; TA, tarsus; TI, tibia; TR, trochanter. Setae and pores present in the first-instar larva of D. sinuosipennis were labeled by comparison with previous papers dealing with the primary chaetotaxy of members of the family Gyrinidae (
The 740 bp COI fragment matching the dataset of
Ultrametric COI gene tree from Beast used to test larvae conspecificity with identified adults using GMYC. Red branches are intraspecific coalecence events, black branches represent divergence between separately evolving lineages. Numbers at nodes are posterior probability values from Beast (only given for species nodes). Sequences from the four larvae are conspecific with adults of Dineutus sinuosipennis but heterospecific to adults of D. proximus and D. subspinosus, according to the GMYC results.
Cephalic capsule constricted at level of occipital region (Figs
Dineutus sinuosipennis Laporte, 1840, instar I. 2 Cephalic capsule, dorsal view 3 Cephalic capsule, ventral view. Numbers and lowercase letters indicate primary setae and pores, respectively. Conspicuous additional seta on frontoclypeus indicated by a solid square. Inconspicuous additional setae not labeled. EB: egg burster; FR: frontoclypeus; PA: parietal; TP: tentorial pit. Scale bar: 0.20 mm.
Instar I (Figs
Color. Cephalic capsule and mandibles light brown, antennae, maxillae and labium testaceous; thoracic sclerites light brown, rest of thorax and legs testaceous; abdomen testaceous except terminal hooks light brown.
Body. Elongate, parallel sided, head and pronotum strongly sclerotized, rest of thorax and abdomen soft. Measurements and ratios that characterize the body shape are shown in Table
Measurements and ratios for the larvae of Dineutus sinuosipennis Laporte, 1840.
Measure | Instar I (n = 2) | Instar II (n = 3) | Instar III (n = 3) |
---|---|---|---|
TL (mm) | 5.50 | 14.80 | 20.10–28.30 |
MW (mm) | 0.70 | 2.20 | 2.40–3.30 |
HL (mm) | 0.87–0.91 | 1.38–1.42 | 2.02–2.05 |
HW (mm) | 0.65–0.66 | 0.99–1.01 | 1.42–1.51 |
FRL (mm) | 0.51–0.55 | 0.78–0.81 | 1.14–1.19 |
OCW (mm) | 0.28–0.30 | 0.55–0.60 | 0.94–0.99 |
COL (mm) | 0.36 | 0.59–0.60 | 0.84–0.88 |
HL/HW | 1.34–1.37 | 1.37–1.43 | 1.34–1.43 |
HW/OCW | 2.20–2.30 | 1.64–1.79 | 1.49–1.53 |
COL/HL | 0.39–0.41 | 0.43 | 0.41–0.44 |
FRL/HL | 0.59–0.61 | 0.57 | 0.56–0.59 |
A/HW | 1.07 | 0.97–1.01 | 0.83–0.90 |
A1/A3 | 0.24–0.25 | 0.24–0.29 | 0.28–0.30 |
A2/A3 | 0.71–0.83 | 1.07–1.15 | 1.28–1.43 |
A4/A3 | 0.96–1.04 | 0.80–0.88 | 0.73–0.74 |
MNL/MNW | 2.94–3.21 | 3.05–3.12 | 2.85–2.93 |
MNL/HL | 0.49–0.53 | 0.45–0.47 | 0.43 |
A/MP | 1.34–1.36 | 1.22–1.40 | 1.25–1.28 |
GA/MP1 | 1.00–1.04 | 0.77–0.84 | 0.65–0.72 |
PPF/MP1 | 0.50–0.55 | 0.48–0.50 | 0.31–0.36 |
MP1/MP2 | 0.65–0.75 | 0.88–0.96 | 1.19–1.30 |
MP3/MP2 | 1.38–1.59 | 1.15–1.26 | 1.06–1.12 |
MP/LP | 1.22–1.29 | 1.23–1.28 | 1.28–1.31 |
LP2/LP1 | 1.16–1.20 | 0.84–0.89 | 0.70–0.73 |
L3 (mm) | 1.74–1.85 | 2.86–2.88 | 4.72–4.75 |
L3/L1 | 1.13–1.15 | 1.21 | 1.26–1.27 |
L3/L2 | 1.06–1.07 | 1.08–1.10 | 1.11–1.12 |
L3/HW | 2.68–2.78 | 2.86–2.91 | 3.12–3.35 |
L3 (CO/FE) | 1.00–1.05 | 1.03–1.07 | 1.08–1.09 |
L3 (TI/FE) | 0.61–0.67 | 0.56–0.57 | 0.54–0.58 |
L3 (TA/FE) | 0.90–0.92 | 0.78–0.84 | 0.66–0.70 |
L3 (CL/TA) | 0.48–0.49 | 0.36–0.41 | 0.35 |
MH/LH | 1.02–1.05 | 1.04–1.06 | 1.07–1.17 |
Head. Cephalic capsule (Figs
Dineutus sinuosipennis Laporte, 1840, instar I. 4 Right antenna, dorsal view 5 Left antenna, ventral view 6 Right mandible, dorsal view 7 Right maxilla, dorsal view 8 Left maxilla, ventral view 9 Labium, dorsal view 10 Labium, ventral view. Numbers and lowercase letters indicate primary setae and pores, respectively. Additional setae indicated by solid squares (except for minute additional setae on the mandible which are not labeled). Additional pore on prementum indicated by a solid triangle. AN: antenna; LA: labium; MN: mandible; MX: maxilla; SP: spinulae. Scale bars: 0.10 mm.
Thorax. Long, narrow, subcylindrical; pronotum somewhat larger that subequal meso- and metanotum; protergite well developed, covering whole segment dorsally, anterior margin truncate, lateral and posterior margins rounded; membrane between pro- and mesonotum with a single narrow transverse sclerite; both sclerites with sagittal line, lacking anterior transverse carina; meso- and metaterga lacking sclerites; ventral surface membranous except for a large subrectangular sclerite (divided in two halves by broad sagittal line) on anterior third of prothorax, and small sclerites on the regions of articulation of coxae; spiracles absent. Legs (Figs
Abdomen. Ten-segmented, long, narrow, subcylindrical, entirely membranous; segments I–VIII similar in shape, progressively smaller to apex, bearing a tracheal gill on posterolateral angle; segment IX smaller than segment VIII, bearing two tracheal gills on posterolateral angle; tracheal gills slender, plumose, those of segment IX longer than the others; all tracheal gills bearing an anterior and a posterior row of long setiform spinulae, those of segment I and, to a lesser extent of segment II with less spinulae; segment X (Fig.
Dineutus sinuosipennis Laporte, 1840. 11–15 Instar I 16 Instar III 11 Left metathoracic leg, anterior view 12 Right metathoracic leg, posterior view 13 Abdominal segment X, ventral view 14 Medial hook, lateral view 15 Lateral hook, lateral view 16 Head, dorsal view. Numbers and lowercase letters indicate primary setae and pores, respectively. Additional pore on trochanter indicated by solid triangle. Sensilla on abdominal segment X not labeled. CO: coxa; FE: femur; PT: pretarsus; TA: tarsus; TI: tibia; TR: trochanter. Scale bars: 0.15 mm (11–15); 0.70 mm (16).
Chaetotaxy. Frontoclypeus (Fig.
Instar II
As instar I except for the following features:
Color. Frontoclypeus, region of parietals posterior to frontoclypeus, and neck region brown, rest of cephalic capsule testaceous to light brown; mandible brown, rest of head appendages testaceous to light brown; protergite with a large subtriangular brown macula on anteromedial region.
Body. Measurements and ratios that characterize the body shape are shown in Table
Head. Cephalic capsule. Occipital suture weakly delimited; rugosity on neck restricted to area of occipital suture; egg bursters absent. Antenna. About as long as HW; A2 the longest, A3 slightly shorter than A2, A4 slightly shorter than A3. Maxilla. MP1 and MP2 subequal in length, somewhat shorter than MP3. Labium. LP1 somewhat longer than LP2.
Thorax. Ventral sclerite of prothorax lacking sagittal line. Legs. Posterior claw shorter than anterior claw on all legs.
Abdomen. Tracheal gills of segment I almost devoid of spinulae, those of segment II with few spinulae.
Chaetotaxy. Cardo with 7–10 short hair-like secondary setae; secondary leg setation detailed in Table
Instar III (Fig.
As instar II except for the following features:
Color. Same pattern but darker and more obvious in general.
Body. Measurements and ratios that characterize the body shape are shown in Table
Head (Fig.
Abdomen. Spiracles present on dorsolateral margin of segments I–III.
Chaetotaxy. Cardo with 8–10 short hair-like secondary setae; secondary leg setation detailed in Table
Number and position of secondary setae on the legs of larvae of Dineutus sinuosipennis Laporte, 1840. Numbers between slash marks refer to pro-, meso-, and metathoracic legs, respectively. A: anterior; D: dorsal; P: posterior; V: ventral.
Segment | Position | Instar II (n = 3) | Instar III (n = 3) |
Coxa | A+AD | 13–16 / 14–15 / 11–18 | 20–26 / 19–25 / 22–27 |
PD | 5–9 / 6–7 / 6–8 | 7–9 / 6–11 / 7–9 | |
Femur | AV | 4–7 / 6–7 / 6–8 | 4–5 / 6–8 / 8–9 |
Tibia | AV | 2–3 / 3–4 / 4–5 | 3–4 / 4–5 / 4–5 |
Tarsus | PV | 5–6 / 5–6 / 6–7 | 5–6 / 6–7 / 7 |
Specimens of D. sinuosipennis were collected on November 28th 2014 in the Betsabora River at crossing of Route National 2, Madagascar (Figs
Very few other aquatic beetle adults (notably no adult D. sinuosipennis) but many juvenile aquatic insects (such as odonates) were found in this habitat, and it was the only place where larval D. sinuosipennis were collected in abundance during the 2014 Madagascar expedition. Given the lack of adult D. sinuosipennis, the abundance of its larval form, and lack of similar numbers of larval specimens encountered elsewhere, such fast-flowing, reedy habitats may represent a preferred larval habitat. This type of habitat is easily over-looked, and may explain why ample larval specimens of one of Madagascar’s most common Dineutus species have not been previously collected or described.
The key was constructed for all instars. Although larvae of Enhydrus were examined only as instar I, the selected character likely applies also to later instars. Larvae of Porrorhynchus are unknown and could not be included. It is likely that larvae of this genus key to the closely related genus Dineutus. Known distribution of Porrorhynchus comprises southeast Asia from southern China to the greater Sunda Islands and west to Myanmar; also in Sri Lanka (
1 | Anterior margin of frontoclypeus lacking teeth (Fig. |
Orectochilini |
– | Anterior margin of frontoclypeus with more or less well developed teeth (Figs |
2 |
2 | Stipes with a series of small hook-like setae on internal margin (Fig. |
Gyrinini |
– | Stipes lacking a series of small hook-like setae on internal margin (Figs |
3 |
3 | Lacinia with posterior margin dentate and apex not deeply indented (Fig. |
4 |
– | Lacinia with posterior margin not dentate and apex deeply indented (Fig. |
5 |
4 | Neck constriction present (Fig. |
Subgenus Andogyrus |
– | Neck constriction absent (Fig. |
Subgenus Macrogyrus |
5 | Tracheal gills devoid of spinulae (Fig. |
Enhydrus |
– | Tracheal gills bearing two rows of long spinulae (Fig. |
Dineutus |
Larval structures (instar I). 19–20 Cephalic capsule, dorsal view 21–26 Anterior margin of frontoclypeus, dorsal view 27–29 Stipes, lacinia and galea, dorsal view 30–31 Tracheal gills, dorsal view 19Macrogyrus (Andogyrus) seriatopunctatus (Régimbart, 1883) 20Macrogyrus (Macrogyrus) oblongus (Boisduval, 1835) 21 Gyretes sp. 22M. (A.) seriatopunctatus23M. (M.) oblongus24 Enhydrus sulcatus (Wiedemann, 1821) 25 Dineutus sinuosipennis Laporte, 1840 26 Dineutus sp. (specimen from Bayfield County, Wisconsin, USA) 27 Gyrinus monrosi Mouchamps, 1957 28M. (M.) oblongus29 D. sinuosipennis 30 E. sulcatus 31 D. sinuosipennis.
The description provided here adds Dineutus to the group of gyrinid genera for which the ground plan of primary chaetotaxy has been described. Considering this description, within the tribe Dineutini only the larvae of the genus Porrorhynchus remain unknown.
Dineutus larvae bear the characters considered as putative autapomorphies of Gyrinidae by previous authors (
Compared to those of the other known Dineutini genera, larvae of Dineutus can be distinguished by the lacinia not dentate on posterior margin (= Enhydrus, dentate in Macrogyrus); tracheal gills plumose (= Macrogyrus, not plumose in Enhydrus); parietal seta PA5 inserted relatively far from setae PA7–9 (closer to PA7–9 in Enhydrus and Macrogyrus); mandibular pores MNb and MNc inserted relatively far from each other (closer to each other in Enhydrus and Macrogyrus); and tarsal seta TA1 inserted submedially (inserted distally in Enhydrus and Macrogyrus). Information regarding these characters in other Dineutini species is scarce, since the larvae of most species are unknown. Therefore, their phylogenetic significance remains to be tested when larvae of more species are studied.
We thank Helena Shaverdo and Cesar Benetti for their valuable comments to the manuscript. MCM was supported by Agencia Nacional de Promoción Científica y Tecnológica under Grant PICT–2014–0853 and by Universidad de Buenos Aires under Grant UBACyT–20020150100170BA. GTG was supported by a NIH IRACDA postdoctoral fellowship (5K12GM064651). Part of this project was funded by NSF #DEB–1402446. We thank Niclas Gyllenstrand at the Centre for Genetic Identification (