Horismenus camobiensis (Hym.: Eulophidae), a new hyperparasitoid of Cotesia invirae (Hym.: Braconidae) in Opsiphanes invirae (Lep.: Nymphalidae) pupae

Horismenus camobiensis sp. nov. (Hymenoptera: Eulophidae), is described based on morphological, molecular and ecological data; this new species of chalcid wasp acts as hyperparasitoid of Opsiphanis invirae (Hübner, 1818) (Lepidoptera: Nymphalidae) in its parasitoid Cotesia invirae Salgado-Neto and Whitfield, 2019 (Hymenoptera: Braconidae). Diagnoses with morphological and molecular characters and illustrations are provided.


Introduction
Horismenus Walker, 1843, with about 420 species, is one of the largest Eulophidae groups and its species act as primary or secondary parasitoid of insects and arachnids (Noyes, 2018;Morales-Silva et al., 2019), mainly in tropical America (Hansson, 2009). Most records mention Horismenus as primary parasitoids, but at least 14 of its species act as secondary parasitoids of Braconidae, mainly of Microgastrinae species (Hansson et al., 2014;Pikart et al., 2015). Thirty-nine species of Horismenus are reported in Brazil (Hansson, 2009;Hansson et al., 2014;Pikart et al., 2015).

Molecular identification
Horismenus camobiensis sp. nov. was characterized by sequencing the mtDNA COI gene of seven specimens from Brazil. The consensus sequences from Brazil showed one SNP (Single Nucleotide Polymorphisms) located at 280 bp from the alignment between the cladogram data sets and identified as a pyrimidine substitution (T/C). The NCBI/Genbank deposit generated the accession number MK455796 for Brazil. The cladogram ( Figure 1) was reconstructed, based on analyses of the COI region performed by the General Time Reversible (GTR) nucleotide substitution model, with gamma distributed with invariant sites; parameters for partial exemption (95%) were estimated as the best substitution model using MEGA 5.0 software (Tamura et al., 2011). The largest possible number of comparatives accessed from those deposited in NCBI was included to perform the cladogram analyses.

Material and Methods
The studied Horismenus specimens were reared from C. invirae, which in turn parasitized O. invirae caterpillars on palm trees (Salgado-Neto and Di Mare, 2010;Salgado-Neto, 2013;Salgado-Neto et al., 2019). Specimens were examined through a Leica M165C stereomicroscope (Leica Microsystems GmbH, Wetzlar, Germany). Color images were taken with a Leica DFC 420 digital camera attached to this Leica stereomicroscope and illuminated with a LED dome (Kerr et al., 2008). The final extended-focus image was combined with Leica Application Suite v3.8. SEM micrographs of uncoated holotype and paratype specimens were taken with a Quanta 250 scanning electron microscope (FEI Company, Hillsboro, USA), at low vacuum mode.
The molecular-specific characterization of the new species was based on its mitochondrial gene Cytochrome Oxidase I (COI). A fragment of approximately 460 bp of this gene was amplified with the primer pair Figure 1. Cladogram of the studied species of Horismenus (Hymenoptera: Eulophidae) inferred from partial mtDNA COI gene region sequences, using Maximum Likelihood analysis. and evolutionary history inferred with the Maximum Likelihood Method (Tamura and Nei, 1993).
The molecular-specific characterization of the new species was performed. A fragment of approximately 460 bp of this gene was amplified with the primer pairs COI-F (5'-GATTTTTTGGKCAYCCMGAAG-3') and COI-R (5'CRAATACRGCTCC TATWGATAA WAC-3') (Gusmão et al., 2010). The DNA of this fragment was extracted from one specimen with the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich®), following the manufacturer protocol. The product was amplified via PCR according to the following schedule: 94 °C for two minutes, 40 cycles of 94 °C for 30 seconds each, 54 °C for 30 seconds, 72 °C for 40 seconds and 72 °C for four minutes. The PCR product was purified using polyethylene glycol precipitation (PEG) (Schmitz and Riesner, 2006). These samples were sequenced using the Big Dye 3.1 reagent (Life Technologies®) and 3500 xL automatic sequencer (Life Technologies®).
Morphological terms used here are from Hansson (2009) and the Neotropical Eulophidae (2018)
Female. Scape yellowish-white, pedicel and flagellum brown, with metallic blue tinges. Frons and clypeal area metallic blue with green tinges near toruli. Vertex metallic blue with green tinges before lateral ocelli, and metallic green with blue tinges posteriorly. Mesoscutum, scutellum, dorsellum and coxae metallic blue with green tinges; fovea of dorsellum and petiole dark metallic purple. Propodeum with smooth parts metallic blue with green tinges and the reticulate ones metallic dark purple. Femora, tibiae and tarsi yellowish-white. Wings hyaline. Anterior 2/3 of the first tergite of gaster with metallic blue with green tinges, the posterior 1/3 and the remaining tergites metallic dark purple. Clava with two segments weak, 1.1-1.2x as wide as the width of the first flagellomere (Figures 3c, 3d). Frons (just above frontal suture, between antennal scrobes and below level of toruli), corners of the mouth and temple smooth and shiny; remaining parts of frons with raised and strong reticulation; frontal suture V-shaped, complete, nearly reaching the eyes; antennal scrobes joining on frontal suture. Vertex with engraved and weak reticulation, with a sulcus coming from behind each lateral ocellus and extending ahead of them, nearly parallel to eyes, until the level of median ocellus; posterior part with a median groove. Occipital margin rounded. Mesoscutum with raised and weak reticulation, posterior half of midlobe with engraved and weak reticulation; notauli indistinct or distinct posteriorly. Scutellum with mesh-rows and engraved and weak reticulation. Dorsellum convex and smooth anteriorly with two foveae connected in the middle. Propodeum smooth and shiny, posterior quarter of submedian grooves and median carina reticulate; propodeal callus with two setae. Coxae smooth and shiny. Forewing speculum closed below, with 13 admarginal setae.    notauli, one should run both ways. If the mesoscutum has narrow and distinct notauli in the posterior half, then H. camobiensis would run to H. iangauldi in couplet 20; but in H. camobiensis sp. nov. has: a. the midlobe of the mesoscutum with engraved reticulation on its posterior part (vs. raised reticulation in H. iangauldi); b. scutellum with strongly engraved reticulation (vs. weakly engraved reticulation in H. iangauldi); c. female with scrobes joining on the frontal suture (vs. females with scrobes joining separately on the frontal suture in H. iangauldi); d. female vertex with a sulcus from behind each lateral ocellus to ahead of the ocelli (vs. this sulcus is absent in H. iangauldi), and e. male scape completely white (vs. completely brown in H. iangauldi). If the notauli are indistinct posteriorly, then H. camobiensis would run to H. amadeus in couplet 34, and in this case the differences between these two species are: a; antennal scrobes joining on the frontal suture (vs. the antennal scrobes join the frontal suture separately in H. amadeus); b. female frons with metallic blue with green tinges near the toruli (vs. metallic dark purple in H. amadeus); c. the female petiole 0.7x as long as wide (vs. 1.4-2.0x as long as wide in H. amadeus); and d. female vertex with a sulcus coming from behind each lateral ocellus and extending ahead of the ocelli (vs. this sulcus is absent in H. amadeus).
Horismenus opsiphanis Schrottky, 1909 also uses C. invirae as host (Salgado-Neto and Di Mare, 2010). However, H. opsiphanis can readily be separate from H. camobiensis sp. nov. by: a. the first tergite of the gaster in has round punctures or weak reticulation in the Male. Similar to female, except as follow: frons bright metallic green with blue tinges. Vertex bright metallic green with blue and golden tinges. Mesoscutum bright metallic green with blue tinges. Scutellum bright metallic green with blue tinges more intense than in the mesoscutum (Figure 3b). Antenna as in Figure 5a. Scape: 2.7x as long as it is wide. Antennal scrobes joining frontal suture separately (Figure 5b) Etymology. The specific epithet is a reference to the type locality, Camobi, Rio Grande do Sul state, Brazil; "camobiensis" originates in the native Guarani language, where kãmobi = kã (breast) and mobi (nipple), in reference to the shape of the hills of the region, is associated with the Latin suffix ensis (origin).