Tyraminergic and Octopaminergic Modulation of Defensive Behavior in Termite Soldier

Abstract

In termites, i.e. a major group of eusocial insects, the soldier caste exhibits specific morphological characteristics and extremely high aggression against predators. Although the genomic background is identical to the other non-aggressive castes, they acquire the soldier-specific behavioral character during the course of caste differentiation. The high aggressiveness and defensive behavior is essential for colony survival, but the neurophysiological bases are completely unknown. In the present study, using the damp-wood termite Hodotermopsis sjostedti, we focused on two biogenic amines, octopamine (OA) and tyramine (TA), as candidate neuromodulators for the defensive behavior in soldiers. High-performance liquid chromatographic analysis revealed that TA levels in the brain and suboesophageal ganglion (SOG) and the OA level in brain were increased in soldiers than in pseudergates (worker caste). Immunohistochemical analysis revealed that TA/OA neurons that innervate specific areas, including the mandibular muscles, antennal nerve, central complex, suboesophageal ganglion, and thoracic and/or abdominal ganglia, were enlarged in a soldier-specific manner. Together with the results that pharmacological application of TA promoted the defensive behavior in pseudergates, these findings suggest that the increased TA/OA levels induce the higher aggressiveness and defensive behavior in termite soldiers. The projection targets of these soldier-specific enlarged TA/OA neurons may have important roles in the higher aggressiveness and defensive behavior of the termite soldiers, inducing the neuronal transition that accompanies external morphological changes.

Fig 1. The absolute levels of biogenic amines in soldiers and pseudergates.

Tyramine (A), octopamine (B), dopamine (C) and 5-HT (D) levels in pseudergates (light grey) and soldiers (dark grey) (N = 23 each). The circles indicate the aminergic levels in each termite individual. Box plots show median values (solid horizontal line), 50th percentile values (box outline), 90th percentile values (whiskers), and outlier values (crosses). Asterisks indicate statistical significance (***p < 0.001, **p < 0.01, *p < 0.05 in exact Wilcoxon rank sum test).

Fig 2. Localization of TA-like-immunoreactive (TA-l-ir) and OA-like-immunoreactive (OA-l-ir) neurons.

TA-l-ir neurons in soldiers (A) and pseudergates (B). OA-l-ir neurons in soldiers (C) and pseudergates (D). Left insets indicate the ventral view of SOG. VPM1, 2, 3 are indicated with black, white, grey filled arrowheads, respectively. Right insets indicate images of other individuals. In right inset of (A), white arrowhead is DUM3 neurons. In right inset of (D), white arrowhead is TA/OA2 neurons. Scale bars indicate 500μm in main picture, and 250μm in insets.

Fig 3. Projection pattern of TA-l-ir neurons.

(A~E) Immunostaining using an anti-TA antibody identified single neurons or clusters of immunostained neurons and their fibers. (A) TA/OA1 cluster forms the commissure under the central complex. (B) The corpora cardiaca (CC) is innervated by TA/OA3. Corpora allata (CA). (C) TA/OA6 and TA/OA7 show characteristic projection patterns. (D) TA/OA2 and TA/OA5 share the descending bundle. (E) In the SOG, DUM1 and DUM2 clusters show symmetrical projection patterns with different staining intensities. Scale bars indicate 100 μm. (F, G) Projection pattern of a DUM2-L neuron visualized by intracellular staining. DUM2-L neurons projected mainly into the mandibular nerves and tritocerebrum, and send thin neurites into the maxillary segment of the SOG. (F) Dorsal view of a DUM2-L neuron. Numerous varicosities were observed in the tritocerebrum (arrowheads). Inset indicates the magnified image of the DUM2-L projection area in tritocerebrum. Scale bars indicate 500μm in main picture, and 100μm in insets. (G) Lateral view of a DUM2-L neuron in the SOG. The neurite extends to the mandibular nerve (white arrowheads) and maxillary neuromere (a black arrowhead). Inset is the image of the different depthplane, indicating the DUM2-L projection into the mandibular nerve. Scale bars indicate 200μm in main picture, and 100μm in insets.

Fig 4. Comparison of soma size distributions of TA/OA-l-ir neurons between soldiers and pseudergates.

(A) Each upper and lower panel indicates the soma size distribution of TA/OA-l-ir neurons in soldiers and pseudergates in the neuronal cluster, respectively (See also Table 3). Dark grey histograms indicate statistical differences between castes. (B) Projection targets of TA/OA-l-ir neurons. The TA/OA neuronal clusters and projection target are indicated as circles and rounded squares, respectively. Neuronal clusters that are soldier- and pseudergate-specifically enlarged are coloured black and white, respectively. Light grey fill indicates no significant differences in the soma sizes between castes. Target regions are subdivided as antennal nerve (Ant N), mandibular nerve (Md N), optic lobe, protocerebrum (Proto), deutocerebrum (Deuto), central complex (C Complex), tritocerebrum (Trito), corpora cardiaca (CC), mandibular segment of SOG (Md seg), maxillary segment of SOG (Mx seg), and thoracic and abdominal ganglia (Thorac/Abd G). The target regions of the soldier-enlarged neurons are filled in dark grey.

Fig 5. Functional analyses of TA and OA application in pseudergates.

(A) Defensive behavior of soldiers and pseudergates anaesthetised but uninjected as controls (N = 10 each). (B) Defensive behavior of TA-treated pseudergates and controls (N = 31 each). (C) Defensive behavior of OA-treated pseudergates and controls (N = 20 each). The circles indicate the biting frequency or duration of orientation in each termite individual. Box plots show median values (solid horizontal line), 50th percentile values (box outline), 90th percentile values (whiskers), and outlier values (crosses). Asterisks indicate statistical significance (**p < 0.01, *p < 0.05 in exact Wilcoxon rank sum test).