The first record of Omosita nearctica Kirejtshuk (Coleoptera, Nitidulidae) in South Africa, with the first description of its mature larva

Sap beetles of the genus Omosita Erichson are stored-product pests that are also associated with carrion, potentially making them biosecurity risks and forensic tools. The discovery of a specimen of the Nearctic species Omosita nearctica Kirejtshuk in South Africa prompted an investigation a decade later to determine if this species had established itself in the country, which was confirmed by the collection of further breeding specimens that also facilitated the first description of mature larvae of O. nearctica. A new key to adults of all Omosita species is presented.


Introduction
Many insects associated with stored products have been moved between continents following humans' colonisation of new places. In the case of cryptogenic species, this invasion process has been so thorough that the geographical origin of the insect invaders is no longer clear, e.g. the Hide Beetle, Dermestes maculatus DeGeer, 1774 (Coleoptera: Dermestidae) (Mroczkowski 1968). In other cases, these invasive species are not noticed for years even though they may be well-known pests in other countries, e.g. the Oriental Latrine Fly, Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) in South Africa (Williams and Villet 2006;Badenhorst and Villet 2018). Insects, and particularly beetles (Midgley et al. 2009), associated with stored animal products, are often of significance in forensic entomology and biosecurity, and keeping track of new members of the carrion insect community in a particular country is important in both of these contexts.
The sap beetle family Nitidulidae has approximately 350 genera and over 4500 species (Lee et al. 2020). The Nitidulidae in Africa have not been well studied and so what is known of them is limited (Kirejtshuk 2001). There are seven recognised species in the genus Omosita Erichson, 1843-O. discoidea, O. colon, O. depressa, O. funesta, O. smetanai, O. japonica and O. nearctica (Reitter 1873, 1874Kirejtshuk 1987;Lee et al. 2015). A specimen of O. discoidea in the Naturhistoriska Riksmuseet, Stockholm * , was collected by Gustav de Vylder (Lee et al. 2015), probably during his stays in Cape Town in 1871-1873and 1879-1885(de Vylder 1998; no other published records of Omosita in the Afrotropical Region were found. At least some sap beetles of the genus Omosita are relevant in forensic entomological and biosecurity contexts because they are occasional pests of stored products and can be abundant on carrion and corpses (Hinton 1945;Shubeck et al. 1977;Jelínek 1999;Kočárek 2003;Ewing and Cline 2005;Schlechter 2008;Saloña et al. 2010;Lee et al. 2015;Lyu et al. 2016;Torres et al. 2018;Lee et al. 2020). Several Omosita species have been translocated around the globe, e.g. the Palaearctic species Omosita colon (Linnaeus, 1758) and Omosita discoidea (Fabricius, 1775) have been reported from Australia and New Zealand (Blackburn 1903;Carlton and Leschen 2007); O. colon has been recorded on Pitt Island, 800 km east of New Zealand (Alfken 1904;Emberson 1998); and the Mexican species Omosita funesta Reitter, 1873 is reported from Spain (Audisio 1990). Despite their applied significance and widespread distribution, literature about the distribution of Omosita species is demonstrably scattered.
Omosita nearctica Kirejtshuk, 1987 was described from North America (Kirejtshuk 1987), but nothing has yet been published about its biology. Some North American records of O. colon published or identified before 1987 may actually represent O. nearctica, which was only recognised as a separate species in that year (Kirejtshuk 1987).
The collection of a single specimen of Omosita nearctica in August 2001 in South Africa suggested the introduction of O. nearctica to this country. This paper reports this discovery, confirms the breeding of O. nearctica in South Africa, and provides the first description of its larva.

Specimen collection
An adult specimen of Omosita nearctica was collected in a trap baited with 50 g of fresh chicken liver in Makhanda (formerly Grahamstown), Eastern Cape province, South Africa, in August 2001, during a study of the seasonal distribution of forensically important flies (Villet et al. 2017;Williams and Villet 2019). The specimen was discovered in 2012 among the ethanol-preserved flies. It was mounted and deposited in the Albany Museum, Makhanda, South Africa (specimen number AM 66416 ** ).
Cooked sheep shank bones were placed in custom-made traps hung about 50 cm above ground in trees at municipal rubbish dumps (or landfills) in Makhanda (33.291°S, 26.492°E) in February 2012 and 2013, and nearby Port Alfred (33.568°S, 26.879°E) in February 2013. The traps were checked regularly and when beetles were caught, they were taken back to Rhodes University and caged with uncooked beef shin bones. Larvae were discovered feeding on the fatty bones in March 2013. The adults (unsexed) and larvae were preserved in 96% ethanol. Five larval specimens were deposited in the wet collection of the KwaZulu-Natal Museum, Pietermaritzburg, South Africa (specimen number NMSA-COL 1405-1409). Adult specimens were card mounted and two specimens were deposited in the KwaZulu-Natal Museum (specimen number NMSA-COL 1898 and NMSA-COL 1410), two in the South African National Collection of Insects, Pretoria, South Africa (accession number SANC-COLG-00021) and two in the Albany Museum, Makhanda, South Africa (specimen numbers AM 101483 and AM 101484).

Identification
The adult beetles (n = 28) were identified from their morphology using the keys in Jelínek (1999) and Lee et al. (2015) and the description and illustrations in Kirejtshuk (1987). A new diagnostic key to the adults of the seven accepted species of Omosita is presented in Appendix 1.
One hind leg of a single beetle (NMSA-COL 1898) was used for DNA analysis. DNA was extracted using the Qiagen DNeasy tissue kit (Qiagen, Inc., Valencia, CA) ** This specimen is misidentified on the Global Biodiversity Information Facility (GBIF) as Omosita japonica (Gess and Ranwashe 2017).
To facilitate comparative biology, a molecular phylogeny of four of the seven species of Omosita was estimated. Additional COI sequences of the four widespread Omosita species were downloaded from the Barcode of Life Data System v4 (BOLD) ( Table 1) and analysed together with the new sequence. Brachypeplus glaber LeConte (Nitidulidae: Cillaeinae) and two species of Nitidula Fabricius (Nitidulidae: Nitidulinae) were used as outgroups. Bayesian inference analyses were performed with Mr-Bayes (Huelsenbeck and Ronquist 2001) using the best-fitting nucleotide substitution mode (GTR+G) from jModelTest (Posada 2008). One cold and three hot chains were run for 5 000 000 generations, sampling every 1 000 generations with burn-in of 1 000 samples (20%).

Larval morphology
Three mature larvae were prepared for scanning electron microscopy (SEM) by criticalpoint drying and sputter-coating with gold (NMSA-COL 1402). The specimens were viewed with a Zeiss Evo LS 15 SEM at the University of KwaZulu-Natal's Microscopy and Microanalysis Unit, Pietermaritzburg, South Africa. Two mature larvae were slide mounted using standard protocols and viewed using a Leica compound microscope (NMSA-COL 1403 and 1404). A further five mature larvae were examined using a Leica dissecting microscope (NMSA-COL 1405-1409). Measurements were taken using a graduated eye-piece.

Morphological identification
Twenty-eight adult specimens of Omosita were collected in Makhanda (1 in 2001, 12 in February 2012 and 15 in February 2013) but none in Port Alfred. The beetles keyed out as Omosita colon using the keys to Palaearctic species of Omosita presented by Jelínek (1999) and Lee et al. (2015), but these keys necessarily do not include O. nearctica, which is as yet unknown from the Palaearctic. Kirejtshuk (1987) Fig. 2).

Molecular identification
The partial COI sequence from one specimen (Genbank accession number: MT371766, NMSA-COL 1898) was 656 bp long and aligned easily with the other sequences. It had a 100% BLAST match to O. nearctica, with the highest match to O. colon at 89.31% (Table 2). In the Bayesian inference tree (Fig. 2), the new sequence forms a clade exclusively containing other O. nearctica sequences.

Diagnosis of adult
Body length 2.4-3.7 mm, oblong ovate, sparsely pubescent, testaceous except for piceous markings on anterior half of elytra, and pale markings on lateral pronotal margins and posterior half of elytra; antennal club not longer than wide; pronotum transverse, concave anteriorly and arcuate laterally, with sides converging more apically than basally, with two oval depressions before scutellum; elytra jointly at least 0.75 as wide as their length, their apices obliquely rounded, forming a common arc and usually exposing one abdominal tergite (Fig. 1). The phallobase is subparallel, with the parameres fused and not divergent; the tegmen is anteriorly transverse and shallowly excavate apically. Omosita colon differs most notably from O. nearctica in the shape of the antennal club which is elongate-oval, much longer than wide and its body shape which is oval. Omosita discoidea differs from O. nearctica in the pronotum colour which is black in the centre and testaceous towards the edges and the antennal club which is longer than wide (Kirejtshuk 1987;Lee et al. 2015)

Description of mature larva
Measurements. Body length 4 mm. Head capsule 0.5 mm wide. Body (Fig. 3). Body campodeiform; subdepressed; widest in abdominal region; white or yellow; uniformly pigmented; poorly sclerotised. Head and all terga with scattered setae; body setae with apices entire.
Head (Figs 4-6). Head capsule (Fig. 4) 2.3 times as wide as long (excluding the labrum); trapezoidal, tapered towards mouthparts; lateral margins straight, at most gently convex; dorsal hind margin slightly retracted; ventral hind margin strongly retracted; granular or tuberculate; with one dorsal and no ventral stemma on each side. Antenna with three antennomeres. Basal antennomere almost as long as wide; without setae. Second antennomere as long as or slightly longer than first; with mesal, subapical sensory area bearing a large cone; with sensory appendix about two thirds the length of third antennomere; with setae, including one directly proximal to sensory area. Third antennomere about as long as basal antennomere; setose with a group of minute apical setae. Frontal sutures impressed, reaching near antennal insertions; frontoclypeal suture distinct laterally, obsolete medially. Clypeus trap-    ezoidal; with three pairs of submarginal setae; clypeal protuberances weak. Clypeolabral suture nearly straight. Mandible (Fig. 5) apex bidentate; with two subequal lateroventral setae. Prostheca consists of several large lobes; bearing a lightly sclerotized projection at base. Mola transversely ridged. Maxilla elongated. Maxillary palp three-segmented; with third joint longer than first or second. Galea with large, dense apical brush. Lacinia partially fused to galea. Mala enlarged inner-distally; bearing rather sparsely scattered microtrichia. Labium about 1.5 times as long as wide. Labial palp one-segmented; set close at base of labium. Ligula strongly produced. Mentum indistinctly separated from submentum. Submentum with two pairs of setae, one proximal and one distal (Fig. 6).

Discussion
This study presents the first record of Omosita nearctica in South Africa and confirms that it is established as a self-sustaining, breeding alien invasive species in the Eastern Cape Province of South Africa. The COI gene (Fig. 2) agreed with the morphological identification of the beetles as O. nearctica. The sequence from this study grouped together in a clade with three other sequences of O. nearctica that were separated from its sister clade, O. colon, with 100% posterior probability (Fig. 2). One sequence (BOLD Public Record COLON045-10.COI-5P) identified as O. nearctica grouped unambiguously with the O. discoidea clade.
The morphological character states listed in Table 3 may be used to differentiate the mature larva of O. nearctica from that of O. colon, the only other species of Omosita for which the larva has been described (Eichelbaum 1903;Verhoeff 1923;Hinton 1945;Böving and Rozen 1962;Hayashi 1978;Díaz-Aranda et al. 2018). The description of the urogomphi of O. colon by Hayashi (1978) differs from the description by Díaz-Aranda et al. (2018). Hayashi (1978) describes them as short and Díaz-Aranda et al. (2018) states they are half the length of the ninth tergite which appears to be more accurate (Fig 9b). It must be noted that the pregomphi and urogomphi are all referred to as urogomphi by Díaz-Aranda et al. (2018). It is crucial to recognise that because O. nearctica was recognised only in 1987, prior references to O. colon larvae from the Nearctic (e.g. Hinton 1945;Böving and Rozen 1962) may be in error.
The collection of adults in Makhanda in 2001, 2012 and 2013 confirmed that O. nearctica has probably established in South Africa. This is important in the global context of this species as it has apparently never been recorded outside North America (GBIF.org 2020). The small size, furtive habits and internationally traded diets of sap beetles in general make them good candidates for transport around the world. For instance, at least 32 extralimital species have established in Europe (Jelínek et al. 2016). Most of these species feed on ripening and decaying fruit, but O. funesta was imported from Mexico to Teruel, Spain in 1931, "probably on imported sausages" (Jelínek et al.  ( Hinton 1945;Böving and Rozen 1962;Díaz-Aranda et al. 2018) were not used because they may involve unwitting misidentifications.

Character
Character state Omosita nearctica Omosita colon Head capsule 2.3 times as wide as long (excluding labrum) 1.4 times as wide as long (excluding labrum) 2 nd antennomere with setae, including one directly proximal to sensory area without setae Sensory appendix of 2 nd antennomere about two thirds as long as 3 rd antennomere about half as long as 3 rd antennomere Mola transversely ridged transversely ridged and asperated Abdominal paratergites touching in midline set fairly close together but not touching in midline Spiracular tubes on A8 as wide as tall wider than tall 2016). Species of Omosita are generally associated with human middens and animal remains (Hinton 1945;Shubeck et al. 1977;Jelínek 1999;Kočárek 2003;Ewing and Cline 2005;Schlechter 2008;Saloña et al. 2010;Lee et al. 2015;Lyu et al. 2016;Torres et al. 2018), so it is less obvious how they were transported to Africa. Omosita nearctica was probably introduced to South Africa on stored products imported through a port or airport. Given the age and remoteness of some austral introductions of Omosita (Blackburn 1903;Alfken 1904;Jelínek et al. 2016), it is possible that the population established well before it was discovered. Their presence in Makhanda (E. Cape, RSA) suggests that they have been in South Africa for many years, since the town has no international airport and the nearest commercial harbours are over 120 km away. The failure to find specimens in Port Alfred (E. Cape, RSA) is ambiguous evidence of the species' distribution because the sampling effort was limited.
Nothing is published about the biology of O. nearctica (Kirejtshuk, 1987). That O. nearctica larvae feed on cooked sheep bones suggests that this species feeds on saponified oils and decomposing material, like at least some other species in its genus (Lee et al. 2015;Lyu et al. 2016). In China, O. colon was the only species of beetle observed breaking down adipocere on corpses, potentially giving that species specific significance in medico-criminal forensic entomology (Lyu et al. 2016). The similar diets and close relationship (Fig. 2) of O. colon and O. nearctica imply that the latter species may be similarly useful (Midgley et al. 2009). Beetles associated with stored animal products are often relevant to biosecurity. Further studies on the biology of this species should monitor its global spread and determine its usefulness in forensic entomology. The description of the mature larva will assist in identifying this species where only larvae are found associated with stored products, corpses or carcasses.