Anatomical asymmetry of the retrocerebral complex in the cricket, Gryllus locorojo (Orthoptera: Gryllidae).
The retrocerebral complex (RCC) is a part of insect neuroendocrine system involved in the control of most aspects of insect physiology. Typically, the RCC consists of paired corpora cardiaca (CC) and paired corpora allata (CA) (Cazal 1948). The; CC consist of axonal endings of neurosecretory cells and their own intrinsic endocrine (glandular) cells and, thus, represent a combined structure functioning as both neurohemal organ and classical endocrine gland (Klowden 2007). Among neurohormones released by the CC are prothoracicotropic (activating the prothoracic glands to produce the ecdvsteroid molting hormone), diuretic, and antidiuretic hormones (neuroparsins, ion transport peptide, and other hormones controlling insect salt and water homeostasis) (Kind el al. 1983, Klowden 2007, Meredith el al. 1996, Schooley el al. 2012, Smith & Rybczynski 2012). Glandular cells of the CC produce adipokinetic and hypertrehalosemic hormones (increasing the lipid and trehalose concentrations in the haemolymph) (Siegert 1999, Schooley e.t al. 2012). The CA are mainly composed of endocrine cells. The main function of the CA is the biosynthesis of juvenile hormone which modulates molt quality (simple or methamorphic) in the larvae and regulates vitellogenesis in the adult insects (Kind el al. 1983, Klowden 2007).
The CC are joined to the cerebrum via nervi corporis cardiaci, the CA are joined to the CC and/or to the suboesophageal ganglion via nervi corporis allati. The left and right CC may be fused in an unpaired structure, as well as the left, and right CA. The variations of this RCC anatomical scheme are characteristics of various insect taxa (Cazal 1948). In Saltatoria e.g., the CC are fused with each other in their caudal parts, the CA are separated from each other, and both allatal nerves joining each corpus allatus with the CC and with the suboesophageal ganglion arc present (Cazal 1948, Gande 1975). A ring gland in higher diptcrans represents the strongest modification of RCC, where the CC and CA are assembled with the prothoracic glands in a single ring-shaped structure (Klowden 2007).
Typically, the RCC is located centrally in the insect body. While working with the cricket Gryllus locorojo Weissman and Gray 2012 (previously reported as "Gryllus argenlinus"), the right side lateralization of the RCC location inside the head capsule was noticed. The aim of the present study was to describe and to statistically confirm this anatomical asymmetry. The confirmation of noticed asymmetry of RCC may be important for the further anatomical characterization of cricket species.
Adult crickets of the species Gryllus locorojo were kindly donated by the Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences (the history and conditions of the year-round culture of these crickets are well described by Zhemchuzhnikov & Knyazev (2012)). Species identification was confirmed using Shestakov & Vedenina (2012) and Weissman el al. (2012).
The crickets were sacrificed by immersion in 70 % ethanol and preserved in the same medium for further use in dissections and asymmetry measurements. The cricket heads were prepared in a Petri dish filled with 70% ethanol using tweezers and dissecting needles under a binocular microscope equipped with a scale and a reticle grid (MBS-9, LOMO, Russia). Heads were oriented dorsal-up for dissection. The dorsal head integument and tissue surrounding RCC were gently removed. To standardize the measurements of RCC location parameters, the heads of the crickets were oriented in a position in which the dorsal and ventral edges of the median ocellus (mo) are visibly merged into a single line (designated in Figure la). Fresh material prepared analogously in an aqueous solution of NaCl (0.75% (w/v)) was used only for general description of RCC location, including the description of asymmetric topography without measurements. Drawings were performed using reticle grid and plotting paper.
The numbers of females and males used for description of the RCC location were 32. Among them, ten adult females were used for detailed measurements of the RCC topography parameters. The primary parameters (Figure 1) were measured using an ocular scale (the value for the smallest scale division was 25 pm at the magnification used). The parameters measured are: sC and dC. the distances from the left and right temples, respectively, to the projection of the caudal (ventral) CC bridge on the line connecting the temples; the sum of them is the length of the line connecting the temples or the maximal head width (Wmax)', sA (dA), the distance from the left (right) temple to the projection of most lateral contour of nearest corpus allatus on the line connecting the temples; the result of subtraction of (sA + dA) from Wmax is the RCC width; sR (dR), the distances from the projection of the medial border of left (right) corpus allatus to the projection of the CC bridge caudal point on the line connecting the temples. On the basis of these primary parameters, the secondary (relative) parameters were calculated and expressed in percents of Wmax: C/Wrnax (s, left; d, right), the sC and dC, related to Wmax: A/Wmax (s, left; d, right), the sA and dA, related to Wmax; R/Wmax (s, left; d, right), the sR and dR, related to Wmax.
Among the methods for studying asymmetry, the comparisons of sizes of left and right parts of anatomical structures as well as calculating the laterality indexes are common approaches to detect leftward and rightward lateralization (Toga & Thompson 2003). In the present study, a relevant approach to detect the direction of CC bridge and whole RCC biases was to compare pairwise the left and right distances measured. Only distribution-free descriptive and comparative statistical procedures were applied to the data collected. The Shapiro-Wilk test (the most powerful normality test according to Razali & Wah (2011)) was used to check the normality of underlying distributions prior to proceeding the description and comparisons. Since a departure from normality was detected (the null hypothesis that the underlying distribution is normal was rejected with the type I error less than 5%), the parametric methods widely used for estimation asymmetry, such as two-way AN OVA. could not be applied (Glantz 1994; Kvam & Vidacovic 2007). The data were presented in the form of median values and quartiles (Me (ql; q3)), and the exact binomial test (the sign z-test) was used in order to test for the null hypothesis that the particular RCC part is equally likely to be biased to the right or to the left side. The applications of binomial tests can be found in Anfora et al. (2011) (in parallel with ANOVA), Arcadi & Wallauer (2011), Rutledge & Hunt (2004) with some specific features depending on the experimental design, the lateralization quality (structural or functional), and the level of lateralization (individual or populational). The binomial approach is used even for much more complicated cases of lateralization, such as in Blois-IIeulin et al. (2012), Leliveld et al. (2010), Palmer (2002).
Calculations were performed in the R-project for statistical computing (www.r-project.org, version 2.13.1). The Bonferroni correction procedure was applied to the p-value taking into account the fact that the measurements of various parameters were perfonned on the same sample (Glantz 1994).
The CC of adult G. locorojo were found to be elongate bodies closely adjoined to the aorta. The CC ventral bridge between their caudal parts is well discernible. The CA are separately located ellipsoid bodies joined to the CC and suboesophageal ganglion via corresponding (allatal) nerves. Thus, the complex consisting of the CC, the CA, and the allatal nerves surrounds the sagittally located pharynx (Figure 2), which is typical for gryllids (Cazal 1948: Gande 1975).
The maximal head width expressed as Me (ql; q3) of adult females was 6050 /mi (5925 /mi; 6138 [micron]m), the relative width of RCC (in % of the maximal head width) was 18.8% (18.7%; 19.1%). The data confirming the rightward lateralized location of CC within the RCC and of RCC elements within the head capsule are presented in Figure 3. The left C/Wmax, A/Wmax, and R/Wmax wrere 53.60% (53.16%; 53.72%), 41.53% (41.34%; 42.38%), and 5.883% (5.750%; 6.233%), respectively. The right C/Wmax, A/Wrnax, and R./Wrnax were 46.40% (46.28%; 46.84%), 39.60% (39.03%; 40.33%), and 1.633% (1.566%; 1.706%), respectively. In each of ten females measured, the left C/Wmax and R/Wmax were larger than their right counterparts, and the true probabilities of rightward lateralization of the CC bridge inside the head capsule and in the whole RCC were not equal to 0.5 (p=0.002 and 0.006, for C/Wmax and R/Wmax, respectively). The left A/Wmax was larger than right A/Wmax in 8 of 10 females measured (in 2 females, the left and right A/Wmax were equal to each other), and the true probability of rightward lateralization of whole RCC inside the head capsule was not equal to 0.5 (p=0.008). Fresh RCC extirpated in 0.75 % NaCl, retains its asymmetric shape after careful dissection. The right-sided bias of CC location inside the head capsule can also be observed through the occipital opening without the preliminary removal of the epicranium. It seems to be a common feature of morphology of both females and males (Figure 4).
The most prominent (rightward) asymmetry in the location of RCC parts was found in case of the CC bridge (as it follows from the comparisons of left C/Wmax and R/Wmax with their right counterparts) while the whole RCC (as expressed by left and right A/Wmax) has almost central position in the head capsule. The most probable explanation of the CC right-sided bias is the right-sided location of the aorta, the head vessel that tightly connects with the medial surfaces of CC. The slightly asymmetric location of CA revealed in most cases can be due to their junction with CC via short nervi corporis allaii I. Since the CC location asymmetry is notable within the unprepared head capsule (Figure 4), the observed feature cannot be explained by preparation carelessness. Also, it needs to be underlined that the CC rightward lateralization is discernible in fresh material (not treated with ethanol), and the ethanol-treated crickets were used just for confirmation of this observation.
Probably, this is the first report of RCC lateralization in crickets. However, considering published literature, it can be suggested that this asymmetry in the relative position of RCC parts is not a unique feature of G. locorojo. The original illustrations in some papers include the figures of distinctly asymmetric RCC (with the biased CC) of house crickets, Acheta domestica (Belyaeva 1964; Neuhauser et al. 1994; Stay el al. 1994; Thomsen 1943) and field crickets, Gryllus campeslris (Cazal 1948) and Gryllus bimaculalus, (Neuhauser et al. 1994) without mentioning the RCC asymmetry in the descriptions. In cases of Acheta domestica in Belyaeva (1964) and Gryllus bimaculatus in Neuhauser et al. (1994), the CC are biased to the right. The other papers contain figures of RCC slices or figures of RCC where it is not clear whether RCC are oriented dorsal-up or ventral-up, therefore, the direction of CC lateralization (rightward or leftward) cannot be distinguished.
No such asymmetry seems to be present in Teleogryllus commodus, judging from figures in Moore & Loher (1988) and Pipa & Moore (1988), but these figures are schematic. If the RCC of Teleogryllus commodus is really symmetric, in context of the phylogenetic relations described for Gryllidae in Weissman cl al. (2012), the rightward lateralization of CC will be considered as symplesiomorphy or homoplasy in species of genera Acheta and Gryllus.
Also, judging from figures in Cazal (1948), the structural asymmetry of RCC can be supposed to be a feature of some species of insect taxa, c> ther than Orthoptera. Right side lateralization of fused CA seems to be present in Stenopsocus irnrnaculatvs (Psocoptera). Antisymmetrically located CA (not fused with each other) may also be found in insects, such as Clonopsis gallica (Phasmida): in this species, according to figure in the same source, the left corpus allatus is located more frontally than the right corpus allatus. Considering this, it is easy to expect the further findings of the various RCC asymmetries and antisymmetries in insect taxa which are not analysed yet.
It is difficult to explain the functional importance of CC and whole RCC asymmetries found in G. locorojo. Apparently, there are no data concerning the differences between the left and right parts of CC related to their neurohemal function, but the specific unequal distribution of stored neurohormones among the loft and right lobes of CC can be a reason of CC bridge anatomical lateralization. It should also be taken into consideration that the RCC connects with other structures, and some of them, such as the aorta, are also asymmetrically located inside the head capsule, as it was mentioned above. The rightward lateralization of more caudal parts of the aorta seems to be very probable, and it can be tested in the future studies. It is not excluded that the location of the CC-aorta complex in crickets may be influenced by anatomical traits of other organs adjoined to it, such as pharynx or salivary glands, and such asymmetries of CC and aorta location might have no functional relevance.
Detailed measurements of CC and CA location may also be performed on the adult male crickets of this species. The RCC glands location in the crickets of pre-adult stages are also of great interest. To test the hypothesis that the RCC rightward asymmetry is a common feature of gryllid anatomy, measurements of topography of CC and CA in other cricket species (primarily of genera Gryllus and Acheta) should also be performed. In conclusion, in the adult cricket, Gryllus locorojo, the CC location within the head capsule and within the RCC is prominently asymmetric, right-side biased. The whole RCC is also right-side biased within the head capsule in most cases. The right side lateralization of CC bridge and whole RCC is confirmed statistically in females (p less than 0.01 in all tests).
This paper has been formed on the basis of my unpublished thesis defended on the Department of Entomology of the St. Petersburg State University in 2001. So I would like to thank the advisor, Dr A.N. Knyazev, for his kind assistance, and Dr A.G. Akimov for introduction to handling the crickets. I!m grateful to reviewers of this paper for their very useful comments including important suggestions which were helpful for me to consider the topic much more deeply. I would also like to thank them and G.O. Kerkeshko and A.V. Khalin for their help in finding the articles cited.
Recibido abril 16, 2013, publicado diciembre, 2013
Anfora, G., E. Rigosi, E. Frasnelli, V. Ruga, F. Trona & G. Vallortigara. 2911. Lateralization in the invertebrate brain: left-right asymmetry of olfaction in bumble bee, Bombus lerrestris. PLoS ONE 6(4), el8903.
Arcadi, A.C. & W. Wallauer. 2011. Individual-level lateralization in the asymmetrical gaits of wild chimpanzees (Pan troglodytes): implications for hand preference and skeletal asymmetry? Behaviour 148, 1419-1441.
Belyaeva, T.G. 1964. Secretion of corpora cardiaca and corpora aliala and their role in the development of house cricket (Gryllus domesticas L.). Zhurnal Obshhej Biologii (Journal of General Biology), 25: 443-452 (in Russian).
Blois-Heulin, C., M. Crevel, M. Boye, k A. Lemasson. 2012. Visual laterality in dolphins: importance of the familiarity of stimuli. BMC Neuroscience 13, 9.
Cazal, P. 1948. Les glandes endocrines retro-cerebrales des insects. Etude morphologique. Bulletin scientifique de la France et de la Belgique. Supplement, 32(1): 1-227.
Gande, II. 1975. Ilistologische Untersuchungen zur Struktur und Funktion des neurosecretorischen Systems der Hausgrille Acheta domesticus L. Zoologischer Anzeiger, 194(3/4), 151-164 (in German with English abstract).
Glantz, S. 1994. Primer of biostatistics (fourth edition). New-York: McGraw-Hill.
Kind, T.V., N.N. Karpunina, k M.S. Tysiachniuk. 1983. Neuroendocrine system and methods of studying its functional activity. Trudy Vsesojuznogo Jentomologicheskogo Obshhestva (Proceedings of the All-Union Entomological Society), 64: 5-28 (in Russian).
Klowden, M.J. 2007. Physiological systems in insects (second edition). New York: Academic Press.
Kvam, P.II. & B. Vidacovic. 2007. Nonparametric statistics with applications to science and engineering. New Jersey: Wiley k Sons, Inc.
Leliveld, L.M.C., M. Scheumann, k E. Zimmermann. 2010. Effects of caller characteristics on auditory laterality in an early primate (Microcebus marinas). PloS ONE, 5(2), e9031.
Meredith, J., M. Ring, A. Macins, J. Marschall, N.N. Cheng, D. Theilmann, II.W. Brock, k J.E. Phillips. 1996. Locust ion transport peptide (ITP): primary structure, cDNA and expression in a baculovirus system. Journal of Experimental Biology, 199, 1053-1061.
Moore, D. k W. Loher. 1988. Axonal projections within the brain-retrocerebral complex of the cricket, Teleogryllus commodus. Cell and Tissue Research, 252(3), 501-514.
Neuhauser, T., D. Sorge, B. Stay, k K.II. Hoffmann. 1994. Responsiveness of the adult cricket (Gryllus bimaculatus and Acheta domesticus) retrocerebral complex to allatostatin-1 from a cockroach, Diploptera punctata. Journal of Comparative Physiology (B), 164(1), 23-31.
Palmer, A.R. 2002. Chimpanzee right-handedness reconsidered: evaluating the evidence with funnel plots. American Journal of Physical Anthropology, 118, 191-199.
Pipa, R. k D. Moore. 1988. Serotonin-immunoreactive neurons in the retrocerebral neuroendocrine complex of the cricket Teleogryllus commodus (Walker) (Orthoptera: Gryllidae) and cockroach Periplaneta americana (L.) (Dictyoptera: Blattidae). International Journal of Insect Morphology and Embryology, 17(4/5), 303-311.
Razali, N.M. k Y.B. Wah. 2011. Power comparisons of Shapiro-Wilk, Kolmogorov-Smirnov, Lilliefors and Anderson-Darling tests. Journal of Statistical Modeling and Analytics, 2(1), 21-33.
Rutledge, R. k G.R. Hunt. 2004. Lateralized tool use in wild New Caledonian crows. Animal Behaviour, 67, 327-332.
Schooley, D.A., F.M. Horodyski, k G.M. Coast. 2012. Hormones controlling homeostasis in insects. In: Insect Endocrinology. 1st edition (L.l. Gilbert ed.). Elsevier, Oxford.
Shestakov, L.S. & V.Yu. Vedenina. 2012. A problem of taxonomic status of "banana cricket" from culture of the Moscow Zoo Insectarium. Entomological Review, 92(3), 262-270.
Siegert, K.J. 1999. Locust corpora cardiaca contain an inactive adipokinetic hormone. FEBS Letters, 447(2-3), 237-240.
Smith, W. k R. Rvbczynski. 2012. Prothoracicotropic hormone. In: Insect Endocrinology. 1st edition (L.L Gilbert ed.). Elsevier, Oxford.
Stay, B., S.S. Tobe, & W.G. Bendena. 1994. Allatostatins: identification, primary structures, functions and distribution. Advances in Insect Physiology, 25. 267-338.
Thomsen, M. 1943. Effect of corpus cardiacum and other insect organs on the colour-change of the shrimp, Leander adspersus. K. Danske Vidensk. Selsk. Biol. Meddel, 19(4), 1-38.
Toga, A.W. & P.M. Thompson. 2003. Mapping brain asymmetry. Nature Reviews Neuroscience, 4 (1), 37-48.
Weissman, D.B., D.A. Gray, H.T. Pham, & P. Tijssen. 2012. Billions and billions sold: pet-feeder crickets (Orthoptera: Gryllidae), commercial cricket farms, an epizootic densovirus, and government regulations make for a potential disaster. Zootaxa, 3504, 67-88.
Zhemchuzhnikov, M.K. & A.N. Knyazev. 2012. Ontogenesis of the cricket Gryllus argentinus Sauss. (Orthoptera, Gryllidae). Entomological Review, 92(2), 146-153.
Alexey V. Razygraev
St. Petersburg State University, 7-9, Universitetskaya naberezhnaya, St. Petersburg, 199034, Russia, e-mail: email@example.com