Chemical analysis of Brasilimeria Stach, 1949 (Hexapoda, Collembola, Neanuridae) hemolymphatic secretion, and description of a new species

Though Collembola is a widespread hexapod its use of chemical compounds for defense has been reported for only a few European species. Chemical composition analyses of the hemolymphatic secretion of Neotropical Collembola using Gas Chromatography with Mass Spectrometry (GC-MS) has been performed for the first time. The GC-MS analysis revealed 32 constituents, such as aliphatic and aromatic hydrocarbons, esters, alcohols, a phenol, an aldehyde and a ketone. Benzyl benzoate, the main component (at 46.98%), is a compound with known acaricide and insecticide properties. This is the first report on chemical constituents produced by Neotropical Pseudachorutinae, genus Brasilimeria, and will permit future secretion comparisons for Collembola. The taxonomic description of the species producing the secretion analyzed is provided; Brasilimeria assu sp. nov. (Collembola, Neanuridae, Pseudachorutinae) is the third known species of the genus; an updated diagnosis of the genus, an identification key, and further remarks on the species Brasilimeria Stach, 1949 are provided.


Introduction
Chemical defense is widespread among terrestrial arthropods but it is especially diverse within Hexapoda [1,2]. In Hexapoda, much attention has been given to insects, due to their extraordinary diversity, ecological, and economic importance. Collembola are basal hexapods, they represent one of the first Pancrustacea groups to conquer land [3]. Collembola have also been studied regarding excretion of chemical deterrent compounds [4][5][6][7]  herein described; Brasilimeria assu sp. nov., was found in the same Brazilian region, a wellknown biodiversity hotspot of the Brazilian Atlantic Tropical Forest. Mass spectrometry (MS) is one of the most popular analytical techniques for chemical composition identification [14,15]. Currently, it is applied to identify "small molecules", (compounds with less than 500Da of weight), isolated from plants [16], fungi [17], algae, [18], animal gland secretions [19], bacteria and microorganisms [20]. Its use also includes diagnostic tests in biological fluids [21], doping tests [22], identification of pesticides or contaminants in the environment [23], and food and beverage quality control [24].
In this context, since there are so few studies identifying and quantifying chemical metabolites in Neotropical Collembola secretions, GC-MS chemical/analytical techniques were considered appropriate to analyze Brasilimeria assu sp. nov., (Collembola, Neanuridae, Pseudachorutinae). A taxonomic description of the new species and an identification key for species of the genus are also provided.

Sampling
Specimens were retrieved from the environment passively; a soil and litter sample was taken from the site, processed in a Berlese funnel for seven days, and the specimens were fixed in ethanol 92˚GL. Hemolymphatic droplets were released through weak spots in the cuticle, also known as reflex bleeding sites (Fig 1).
The samples (droplets) of the Brasilimeria assu sp. nov., secretions were collected directly from the specimen with the aid of a capillary tube, and before being analyzed were kept in a glass vial under refrigeration.

Nomenclatural Acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and the new names contained within the electronic edition of this article are available using this same code. This published work and the nomenclature acts it contains have been registered with ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) are available and the associated information can viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub: 0D70962F-7F24-4EF3-8160-7B145F2C7555. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

GC-MS analysis
Chemical composition analysis of the secretions was performed using a Gas Chromatograph Mass Spectrometer (GC-MS), Shimadzu, model QP 2010. Separation of the chemical constituents was carried out using a DB-5MS column [(5%-phenyl)-methylpolysiloxane] brand Agilent J&W GC Columns, 30 m long, 0.25 mm internal diameter, thickness of the film 0.25 μm. The carrier gas was helium. The gas chromatograph operating conditions were: column internal pressure of 84 kPa, total flow 10 ml min -1 , column flow of gas at 1.4 ml.min -1 ; Linear Velocity 42.7 cm.sec -1 , Purge Flow: 3.0 mL.min -1 , column ion source temperature 250˚C, low vacuum 7.4 Pa, high vacuum 5.9 −4 Pa, Injection Mode: Split-less, detector temperature at the (GC/MS) interface: 320˚C. The initial column temperature was 40˚C for 4 min, followed by increases of 8˚C min -1 up to 320˚C (being then kept constant for 59 min). The split ratio was 5:1. The mass spectrometry was programmed to perform readings in a range of 35 to 600 Da at intervals of 0.25 s, with the ionization EI (electrospray ionization) mode and ionization energy at 70 eV. One μl of secretion was injected (1 drop was dissolved in 2 ml of ethyl acetate). A mixture of linear hydrocarbons (C9-C20, C21-C40) was injected under the same conditions in order to help identify the chemical constituents. The identification of the constituents was based on data libraries; analyzing and comparing mass spectra (FFNSC1.3.lib, WILEY7.LIB, NIST08s.LIB, MY LIBRARY.lib). The GC/MS equipment indices presented similarities of � 90%. Relative quantification of each constituent was obtained from the relative area of the peaks in the chromatogram.
Head: Eyes 6+6, disposed in a semi-circle; pigmentation under each eye, but not in the periorbital; PAO absent yet substituted with shallow tegumentary depression composed of primary granules (Fig 2D). Dorsal head chaetotaxy: pluri-chaetotic and asymmetrical, rows of chaetae not clearly defined (Fig 2F). Labium with chaetae A-G, C and D apically displaced (Fig 2E). Mandibles: long, with about 20 teeth, presenting two strong basal teeth and about 17 smaller apical teeth, with the five most apical teeth longer than the others; maxillae: stylet-like ( Fig  2C).
Etymology. As a reference to one the locations where the specimens were found, "Castelos do Açu", the word from the indigenous Brazilian Tupi language "Açu" Latinized to "assu", which means big, was considered appropriate given the large size of the new species.
Remarks. Analysis of specimens deposited at the Collembola Collection of the Museu Nacional/UFRJ-Rio de Janeiro-Brazil, (two previously known species: Brasilimeria, B. anura and B. wygodzinskyi), revealed important characteristics not mentioned in the original descriptions. In addition to conspicuous coloration patterns that clearly characterize the species, these also presented a distinctive S-chaetae body formula: 22/22211, i.e. besides the 2+2 S-chaetae on Th. II and III they also present 2+2 S-chaetae on Abd. I-III; being one pair on the De chaetae group and another on the DL group. The new species Brasilimeria assu sp. nov., presents neither patches of white or yellow on its dark blue body, nor a pair of DL S-chaetae on Abd. I-III, and the new species presents rather long ordinary body chaetae; with an ordinary chaeta, S-chaeta ratio = 1:4.This remains in contrast to a ratio of at least 1:9 in the other two species (assuming equal length S-chaetae in all three species). We note that none of the analyzed specimens present DL S-chaetae on Abd. I-III, suggesting that the absence of such chaetae; in contrast to what has been observed in other species of the genus the presence of a reduced S-chaetae formula (022/11111) is diagnostic for Brasilimeria assu sp. nov. Two different S-chaetae formulas are proposed as characteristic for the genus, and thus attention must be given to the presence of two pairs of S-chaetae on Abd. I-   III. The formula 22/22211 is thus not restricted to Pseudachorutinae, though in Neanuridae as a whole, it is both remarkable and uncommon.
According to Queiroz & Zeppelini [12] the antennal chaetotaxy of Brasilimeria is similar to that of Handschinurida, which is corroborated in the description of Brasilimeria assu sp. nov. Further, antenna scheme differences encompass a group of 3-4 latero-external (almost ventral), smaller, and slender chaetae similar to S-chaetae of Ant. IV, between S8 and S10. These groups of chaetae were observed on all three species of the genus. Another remarkable feature is the number of chaetae on Ant. I, between 10-12 chaetae, higher than the usual 7 chaetae seen in the Pseudachorutinae genera [26].
An interesting age-related aspect in specimens of this genus regards their mandibles: juveniles, (generally up to 1.5mm), have mandibles with only 10-12 teeth. This was observed in about ten juvenile specimens. In fact, the description of B. anura, Arlé [27] illustrated the mandible of a juvenile with only ten teeth. Afterwards, Cassagnau & Rapoport [28] depicted a B. anura mandible based on specimens from the Tijuca forest in Rio de Janeiro, (its type locality) with twenty teeth. B. wygodzinskyi illustrations in turn, reveal the actual shape of an adult Brasilimeria mandible. This is the first report of mandible size and shape modification within Pseudachorutinae, and perhaps in Collembola as a whole throughout post-embryonic development. Within the genus Brasilimeria however, adult mandible shape and number of teeth is very stable, as verified in all three species.
The genus Brasilimeria is characterized by the absence of furca (ventral abdominal appendages of segment IV which are modified for jumping), and by large adult specimens, being generally longer than 3 mm, and even reaching 4 mm. The species thus presents reduced mobility and little potential for predator evasion, depending entirely on short cursorial legs that result in clumsy movements. In this sense, the large presence of insecticidal and acaricide substances, such as benzyl benzoate, may be considered as a great ecological/evolutionary advantage against predators.
Brasilimeria assu sp. nov., and the two previously known species of the genus, B. anura (Arlé, 1939) and B. wygodzinskyi (Arlé, 1943), have distinctive color patterns: being a dark blue body contrasted by bright yellow or white spots in differing arrangements over the whole body, this with a bright ventral body and appendages.
As pointed out by Bellinger, such color patterns can be an aposematic indication for unpalatability [41]. The Neotropics present many brightly colored and patterned Pseudachorutinae species (see Oliveira & Deharveng [42] for Pseudachorutes Tullberg, 1871 species; and Queiroz & Mendonça [43] for Arlesia Handschin, 1942, and Handschinurida Queiroz, 2015 species). Nevertheless, in order to relate unpalatable or repellent components in the hemolymph to aposematism, additional comparative studies will be required.
The presence of benzyl benzoate, in Brasilimeria assu sp. nov. secretions is primarily acknowledged as a consequence of food ingestion and consequent incorporation to the hemolymph. In addition to other substances, decaying leaves and twigs of plants that synthesize the compound may be ingested by the species. This is corroborated by the distribution of two plant species that produce benzyl benzoate, Myroxylon balsamum (L.) Harms, and S. hirsuta; as well as many species of the genus Solanum [44], which are congruent with that of Brasilimeria assu sp. nov.

Conclusions
The taxonomic description of Brasilimeria assu sp. nov., together with analyses of B. anura, and B. wygodzinskyi revealed for the first time (within Collembola) important post-embryonic modifications in the shape and size of the mandibles, with teeth numbers increasing from juveniles to adults. Other original data involved the presence of ten or more chaetae on (Ant. I) of Brasilimeria, as well as B. anura and B. wygodzinskyi which present two pairs of S-chaetae on (Abd. I-III); being unique features within Neanuridae. Detailed analyses and descriptions of larger species in the tropics, such as those of Brasilimeria, will be needed for a better understanding of Pseudachorutinae taxonomic characters as well as generating reliable evolutionary scenarios and phylogenetic hypotheses.
Analysis of Brasilimeria assu sp. nov., secretions (using GC-MS) permitted detection of 32 constituents. The major component at 46.98% of the sample volume was benzyl benzoate, which is known for its insecticidal action. It represents a defense mechanism against predation.
Our results on the chemical composition of Brasilimeria assu sp. nov., secretion permit future comparisons of metabolites secreted by other Collembola species, and allow conclusions to be drawn regarding both chemotaxonomic classification and evolutionary history.