Larval morphology of the water beetle Ochthebius balfourbrownei (Coleoptera: Hydraenidae) from marine rockpools of Cape Verde Islands

The morphology of larval instars I, II and III of the water beetle Ochthebius ( Cobalius ) balfourbrownei Jäch, 1989 from Cape Verde Islands is described and illustrated for the first time, by scanning electron microscopy, with a special emphasis on their chaetotaxy and porotaxy. Specimens used in this study were collected with adults of the same species in marine rockpools. A detailed description of characters is provided for comparison with the other members of the same genus inhabiting the marine rockpools, and illustrations of the diagnostic features are presented, with the aim of providing information useful for taxonomic and phylogenetic studies of the genus Ochthebius .


Introduction
Ochthebius s.l. is a large genus within the water beetle family Hydraenidae that includes nearly 540 species worldwide (Jäch 1989;Hansen 1991Hansen , 1998Perkins 2007;Villastrigo et al. 2019), generally found at the margins of freshwater habitats, although some species prefer saline or brackish waters, having different degrees of halophilous tendencies (D'Orchymont 1932;Abellán et al. 2009;Perkins 2011;Sabatelli et al. 2016). Among the halophilous species, some Ochthebius are known to live for their entire life cycle in hypersaline ephemerous marine rockpool environments, found on rocky shores worldwide.
Phylogenetic relationships of the Ochthebius s.l., based on adult morphology, have long been the object of debate. Jäch and Skale (2015) divided the genus into four subgenera: Asiobates C. G. Thomson 1859, Calobius Wollaston 1854, Enicocerus Stephens 1829 and Ochthebius (s.str.) Leach 1815. However, a recent phylogenetic study on this group based on molecular data (Sabatelli et al. 2016) depicted a more complex systematics, later confirmed by Villastrigo et al. (2019), that recognised the presence of 10 subgenera within the genus Ochthebius.
A relevant difference between the classifications is that Cobalius Rey (1886) represents an effectively separate subgenus (Sabatelli et al. 2016;Villastrigo et al. 2019). Cobalius currently includes 15 described species (Jäch 1989;Jäch & Delgado 2017;Ribera & Foster 2018;Sabatelli et al. 2018;Ribera & Cieslak 2019;Villastrigo et al. 2020), all living in marine rockpools with the sole exception of Ochthebius (Cobalius) serratus Rosenhauer 1856, associated with saline streams and pans (in southern Spain and northern Morocco). Members of Cobalius are widely distributed in the eastern Atlantic Ocean (British Islands, Atlantic coasts of continental Europe, Cape Verde, Canary Islands, Morocco, Madeira and Azores) and throughout the whole Mediterranean basin. marine rockpools, to discuss the adaptive value and convergent evolution of characters possibly related to this peculiar environment.

Sampling
Ochthebius balfourbrownei (Figure 1(b)) from Cape Verde Islands was found on the island of Sal (Ilha do Sal), in hypersaline pools in shallow depressions on rock surfaces above the high tide line (Figure 1(a)), but within the splash zone. The pools contained variable numbers of the beetles; they were usually found on the submerged rock surfaces of the pools, in fissures or among submerged algal vegetation.
The larvae described below were collected by the first author together with F. Mosconi in February 2014 from two localities of the island of Sal (Murdeira and Buracona), with adults of the same species, and identified ex societate imaginis. They were collected from the pools by hand with soft forceps or, after vigorous agitation of the water, from the water surface.

Examined material
A total of 20 larvae of Ochthebius balfourbrownei were collected: three first instars, seven second instars and 10 third instars. All larvae were initially preserved in 70% ethanol and deposited in the Sapienza University (Rome, Italy) Zoological Collection (MZUR).

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S. Sabatelli et al. Three specimens (1 first instar and 2 third instars) of O. balfourbrownei were rehydrated, cleared in 10% KOH, transferred to hot lactic acid, dehydrated through a series of EtOH baths of increasing concentration (10, 20, 50, 70, 90, 95 and 100%), left overnight in a clove oil bath and mounted on slides with Canada balsam. They were studied and measured using an Olympus BX51 light microscope, equipped with a drawing tube.

First instar
Habitus and colouration Elongate, slender. Head and body sclerites moderately sclerotised. Colour of sclerites uniformly pale brown. Total body length about 1.4 mm (Figure 2(a)).
( Figure 2(b)). Clearly distinguishable from the first instar due to the lack of cephalic egg-bursters (EB). It differs from the third instar by the presence of dorsopleurites and ventropleurites not yet fused to tergi and abdominal sternal. Chaetotaxy identical to first instar.

Discussion
Larvae of Ochthebius (Cobalius) balfourbrownei show different characters present in other larvae of the genus Ochthebius inhabiting marine rockpools (Delgado & Soler 1996, 1997a, 1997bDelgado & Matsui 2000;Sabatelli et al. 2013). Herein, we excluded the larva of O. lejolisii from the comparison, because it was not described with modern and comparable criteria (D'Orchymont 1913). In comparison to the only larva of the Cobalius group to date studied according to modern criteria, O. subinteger (Delgado & Soler 1996), the larvae of O. balfourbrownei show numerous shared characters: presence of campaniform sensillum (FC1); presence of egg-bursters (EB), here extremely reduced; absence of temporal seta T2, a character that so far, as suggested by Delgado and Soler (1996) and Delgado and Archangelsky (2005), could constitute an autapomorphy for Cobalius; absence of campaniform sensillum (LC1) on lateral regions; absence of V2 on ventral regions; absence of tibiotarsal setae Ad1; presence of setae DP1 and DP2 on dorsopleural sclerites; sternite I without setae P1 and P3; absence of additional seta (D2) and pygopod without anal hooks (AH). However, the larvae of O. balfourbrownei may be distinguished from larvae of O. subinteger by the subapical, mesodorsal presence on antennomere II of one campaniform sensillum (IIC1), which seems to be an autapomorphic character of this species; Ligula (Lg) well developed, composed by two globose lobes; presence on mandibles of one conspicuous, triangular premolar tooth, slightly curved towards base; penicillus extremely reduced; molar area with two rows, one dorsal and one ventral, of small, denticulate microsculpture, basally directed; second labial palpomer with very small seta, medially directed; pronotum with one additional small seta posterior to A1 (A); mesonotal campaniform sensilla C1 and C2 absent and sternite I with one discal seta (D1).
On the other hand, some characters that are considered phylogenetically informative are shared with species belonging to Calobius and Ochthebius capicola, which share the marine rockpool habitat with O. balfourbrownei. The following characters are synapomorphic: (1) presence of mandibular penicillum; (2) absence of tibiotarsal setae Ad1; (3) absence of additional seta (D2); and (4) absence of anal hooks. The larvae of O. balfourbrownei may be distinguished from larvae of O. danjo by the different state of all the above-mentioned characters, as shown below, despite both living in the marine rockpools. In conclusion, the results of our study provide additional support to the phylogenetic relationship between O. balfourbrownei and the species belonging to Calobius and Ochthebius. Likewise, this study highlighted the close relationships of O. balfourbrownei and O. subinteger, as confirmed usingthe molecular analyses of Sabatelli et al. (2016) and Villastrigo et al. (2019Villastrigo et al. ( , 2020, with which it shares numerous characters, particularly the loss of temporal setae T2, considered at present an autapomorphic character for the genus Cobalius by Delgado and Soler (1996) and Delgado and Archangelsky (2005).  -Labral marginal setae Lm1 of normal shape; pygopodial setae present; urogomphal campaniform sensilla present; short urogomphal setae U6 ...... 3 3. Cephalic temporal setae T2 present; cephalic ventral setae V2 present; pronotum with one pair of additional setae posterior to A1; meso-and metanotum with one pair of additional setae close to ecdysial line; sternum with one new lateral subprimary seta L' (Sabatelli et al. 2013