Colobopsis anderseni (McArthur and Shattuck, 2001)

Nielsen, Mogens Gissel, 2022, The biology and physiological adaptation of mangrove ants to tidal flooding, Asian Myrmecology (e 015004) 15, pp. 1-20 : 7-14

Colobopsis anderseni

Colobopsis anderseni (McArthur and Shattuck, 2001) , - previously known as Camponotus anderseni - is restricted to Sonneratia alba trees and is the most common of the 10 ant species found on S. alba trees in Darwin Harbor ( Nielsen 2000). Sonneratia alba trees dominate the vegetation zone at the lowest tidal levels, forming a belt ranging from 30 meters wide to single scattered trees. Founding C. anderseni queens gnaw holes in the young green terminal shoots of these trees, and afterwards carve out nest’s cavities, which are later enlarged by daughter workers. Nest cavities extend through an entire internode (mean length 5.2 ± 0.6 cm), but almost never beyond ( Fig. 6 ).

The whole tree is colonised so that the nests in the upper parts are not inundated but the lowest nests, situated 5-6 m above LAT, are flooded for more than 60% of the time at high tide, with up to 2-3 meters of water above the nest. The founding of new nests commences when the tree reaches a height of 6-7 meters above LAT, and often all internodes in the new green shoots contain founding queens. Each tree functions as an island; consequently, the queens and workers have no contact with the neighboring trees.

The yearly growth of S. alba consists normally of three new green shoots from the terminal part of the twig, each containing 1-3 internodes. After a year the shoots turn brown and lignify. The founding queens are entirely restricted to the fresh green shoots for the excavation of new nest chambers. As the excavation continue and the twig grows thicker, the nest enlarges and all life stages can then be found in the cavity. In nest of C. anderseni adult ants, brood, and coccids can fill up to 50% of the cavity volume.

Both queens and large workers can effectively block the entrance holes with their heads when the branches are inundated, preventing seawater entering the nest but also stopping gas exchange ( Fig. 7 ).

The workers C. anderseni are seldom seen outside on the Sonneratia branches and apparently rely almost entirely on carbohydrate secretions and predation on the single species of a large (up to 8 mm long) undescribed Myzolecanium coccid, which is found only in C. anderseni nests ( Nielsen 2000).Apparently, these coccids can disperse independently, possibly by the wind, as well as being transported by worker ants (P. Gullan pers.com.). They are found in almost every inhabited nest chamber (mean number 3.9, range 1-16, N = 225). Two other undescribed coccid species are found in the nests of the other ant species in S. alba trees.

Surveys of more than 600 nests have shown that queens in terminal shoots tend to disappear within a year, until just one old queen, in the first formed chamber at the base of the branch, remains (Nielsen and Birkmose, unpublished data). At that stage the colony consists of only one nest with an egg laying queen and up to 60 nests with just brood, workers, and coccids, but the excavation of each nest was commenced by a colonizing queen. In the 225 nests whose contents were counted, there was on average (with standard error) 15.9 + 14.2 workers, 3.4 + 2.5 larvae, 2.5 + 2.4 pupae, 1.5 + 1.4 alates ( Nielsen 2000).

Nielsen (unpublished data) recorded the temperature (Tinytag datalogger) in the first 16 days in April in the nests of C. anderseni and found that the mean daily maximum temperature +SD inside the nests was 40.1+ 1.6 0 C which was 4.6+1.5 0 C warmer than the air temperature. The highest temperature measured was 43 0 C, which is close to lethal temperature for insects, and the air temperature was only 35 0 C. The air temperature at the nest site in the mangrove was 2.5 0 C warmer than the official temperature for Darwin (Nielsen unpublished data).

When the branches grow and the thickness of the walls in the nest increase, and when the wall reaches more than 10 to 12 mm the coccids cannot reach the phloem and they die. So, to maintain the colony’s food supply, there is a demand for new nest sites with thin nest walls. However, the old queen is confined in her nest because of her swollen gaster and is therefore unable to gnaw holes for new nest sites and the workers are unable to gnaw a hole for themselves. Consequently, the colony will die out if they don’t recruit new queens to excavate new thinwalled nests. Vacated nest cavities can be used by other ant species such as species of Crematogaster , Tetraponera punctulata and Tapinoma spp.

To investigate further why the newly settled queens disappear microsatellite techniques were used to analyse the genetic structure of the colony of nests (Nielsen, Pedersen and Boomsma unpublished data).

Maps of all the nets in four branches were constructed, and from all nests DNA were extracted from workers, sexuals and brood. Three microsatellite loci were used to analyze the genetic structure. (More detailed description of methods in Appendix 2)

The results show clearly, that the workers and brood in the nests with missing queens were genetically different from those in the nest with the remaining dominant queen. It is thought that the missing queens were killed by the workers produced by the dominant queen and the orphaned workers and brood were adopted by the nest with the dominant queen, even though they mostly remained in the home nest. The micro satellite investigation confirmed this but did also identify that a few had moved to other parts of the colony.

Thus, it seems that a colony get new workers from new queens with coccids enabling the founding nest, with the dominant queen, to survive for her full life span. When the old queen does die, she may be replaced by one of the newly established queens on the branch.

Nest gas concentrations and physiological adaptations

The mangrove ants that are subjected to regular flooding by tidal cycles are living in a stressful environment. Not only are they often subjected to high temperatures but, for varying lengths of time, there is no opportunity for gas exchange with the atmosphere. Ants can be tolerant of high temperatures ( Bujan et al. 2020) and have adapted to conditions associated with colonial life such as elevated levels of CO 2 concentration. Fungus growing ants have often highly elevated CO 2 concentration in their nests. Romer et al. (2018) and Kleineidam and Roces (2000) investigated the carbon dioxide concentration in the giant nests of the leaf cutting ant Atta vollenweideri , and found that the carbon dioxide concentration never exceeded 2.8% in the dump area below the fungus garden, and the highest concentration in the nesting area was 1.5%. Sousa et al. (2021) measured the CO 2 concentration in the nests of Atta sexdens and found values up to 5.7 %, whereas the harvester ant Pogonomyrmex badius had concentration 0.2% CO 2. The CO 2 concentration in ant nests in the ground are strongly influenced by the CO 2 concentration in the soil atmosphere, which normally range from 0.3 to 3.0 %, but can exceed 10 % in anaerobic soils ( Lavelle and Spain 2001). The atmospheric concentration is 0.04 %, and even large nests of O. smaragdina have only slightly elevated CO 2 concentration.

During high tide P. sokolova retreats to air pockets in the nest until the water level in the nest drains. During this period the ants and brood only have a limited amount of O 2 available and must cope with the increasing concentrations of CO 2. Measurements of the CO 2 concentration in air samples from nest chambers at different depth shows great variation, from 2% to 11% CO2 ( Nielsen et al. 2003). By comparing the CO 2 concentration from artificial galleries without ants, and the respiration of ants and mud samples, we could estimate that the ants only contributed 10 to 15% of total CO 2 concentration and the remaining from the microbiological respiration in the mud.

The strategy of C. anderseni is quite different to that of P. sokolova and more like that of other arboreal ants in the tidal zone and easier to investigate. Each small nest chamber in C. anderseni colonies is separate from the other chambers and there is only one entrance. Shortly after the excavation, the twig will get woody and all gas exchange takes place through the entrance.

The CO 2 concentration in the nest of C. anderseni were measured in the laboratory from gas samples from the nests in the field. The mean +SD concentration of CO 2 in the nests was 5.5+3.2 % (range 1.5 –12.5%). The very large deviations due to the variations in the content of biomass, where the mean + SD biomass volume in the nests was 22.7 + 9.4 % of the total nest volume (range 1.5 to 49.6 %) ( Nielsen et al. 2006).

The production of CO 2 in whole sealed nests in periods from 0.5 to 240 minutes were measured and the respiratory rates (uL CO 2 per mg per hour) were calculated at the end of the experiments together with the concentration of CO 2. The CO 2 concentration reached extremely high levels, over 30%. At 10 % and 25 % CO 2 the respiratory rate had decreased to 18,9 % and 1.8 % of rates in atmospheric air, respectably ( Nielsen et al. 2006). (More detailed description of methods in Appendix 3)

Hypoxic conditions and oxygen supply in nests of the mangrove ant, C. anderseni , during and after inundation were investigated under seminatural conditions ( Nielsen et al. 2009). A hypodermic needle, with a fiber-optic micro sensor connected to an oxygen meter (PreSens Microx TX3) was inserted into the nest, such that the sensor tip extended 2–5 mm inside the cavity ( Fig. 9 ). The O 2 concentrations inside the nests were measured continuously, and when stabilized, the nest entrance was then sealed. The seals were removed after 1–2 hours when the O 2 concentration had fallen to 4% or less and measurements continued until the values returned to the starting level ( Fig. 10 ).

During normal conditions with open nests, the oxygen level is substantially lower in the parts at greatest distance from the entrance, such that in a 120 mm long nest the oxygen concentration in the air can be as low as 15.7%. During simulated inundation described above, the oxygen concentration dropped to very low levels, sometimes less than 0.5% after one hour. After opening the nest entrance, the oxygen level was restored to the starting level in about 20 minutes in a 100 mm long nest.

A series of experiments were carried out to investigate the rate of decrease in the O 2 concentration in a closed respiratory chamber with C. anderseni . The results show that the O 2 concentration decreases linearly until it is about 18%, then there is an exponential decrease until levels reach 4% upon which the slope becomes much lower ( Fig. 11 ) ( Nielsen and Christian 2007). A similar experiment but with a CO 2 absorber in the chambers found a linear decrease until 4% O 2 which suggests the rate of decline is reduced because of the high concentration of CO 2.

Some simple and preliminary experiments of the tolerance of low O 2 and high CO 2 concentrations were conducted with C. anderseni and, for comparison, the arboreal O. smaragdina . The ants were kept in small glass tubes with moist filter paper in low oxygen or high carbon dioxide concentrations for 72 hours or until all the ants were dead. The preliminary results for the low oxygen levels were that both species could survive in 5% O 2 + 95% N 2. With 3% O 2 + 97% N 2 most O. smaragdina died and it is doubtful that they would survive for long at that concentration in nature. Colobopsis anderseni had a higher survival at 3% O 2 but more detailed investigations are necessary to determine whether they can survive normally in this concentration. In high carbon dioxide concentrations (>30%) all O. smaragdina died within 3 - 4 hours irrespective of the O 2 concentrations, whereas C. anderseni could survive 10-15 hours. With 100% N 2 the survival was the same as 3% O 2 (Nielsen unpublished data). 3 - 5% oxygen concentrations seem to be a critical concentration, when the effect of high CO 2 concentrations is absent.

The remarkable ability that C. anderseni shows to survive extreme atmospheric conditions suggests, that they might switch to anaerobic respiration. A series of experiments were performed to examine this hypothesis ( Nielsen and Christian 2007). About 100-150 ants were placed in small double-necked Warburg chamber, which was connected to a CO 2 analyzer and with a O 2 sensor inserted into the chamber. The experiment lasted for between 15 minutes and four hours. O 2 concentrations were measured continuously, as was the amount of produced CO 2 at the end of the experiment.

The respiratoric quotient RQ is the ratio of CO 2 production and O 2 consumed. The value indicate which macronutrient are metabolized – only lipids give a value of 0.7 and protein and carbohydrate give 0.8 and 1.0, respectively. Values above 1.0 can only occur during anaerobic metabolism or during transformation of carbohydrate to lipids - which is very unlikely (Keisner and Buch 1964). The results clearly showed that anaerobic respiration occurs at levels higher than 18% CO 2 ( Fig. 12 ). This is the first time that anaerobic respiration has been demonstrated in social insects. In a latter series of experiments C. anderseni , P. sokolova and, for comparison, the strictly aerial O. smaragdina were kept under anaerobic conditions and subsequently tested for lactate, the usual end product of anaerobiosis in animals. None produced lactate (Nielsen unpublished data). However, it is known that some insects produce several anaerobic end products besides lactate ( Hoback and Stanley 2001). Alanine is an important end product for leaf beetles Agelastrica alni ( Kolsch et al. 2002) and for aquatic insect larvae a significant end product are arginine, alanine, ethanol and succinate ( Redecker and Zebe 1988), none of which have yet been assessed for ants.

In a further study, the changes in metabolites in these ants were investigated by using nuclear magnetic resonance spectroscopy (MRS) ( Malmendal et al. 2006, Nielsen, Malmendal, and Henriksen unpublished data). Colobopsis anderseni , and O. smaragdina showed the same pattern with an increase of glucose, maltose and alanine and a decrease of coenzyme A and trehalose, whereas P. sokolova showed an increase in alanine and acetate. It is very interesting that the strictly arboreal ant O. smaragdina which never experiences inundation was able to switch to anaerobic respiration. Maybe this capability is universal within ants?

Figures

Fig. 6. The founding queen of Colobopsis anderseni in a newly excavated cavity in a twig of Sonneratia alba. Photo Dorthe Birkmose

Fig. 7. A Colobopsis anderseni soldier blocks the nest entrance to prevent water intrusion. Photo M. G. Nielsen.

Fig. 9. A piece of a Sonneratia alba twig with a nest of Colobopsis anderseni in the laboratory. The nest entrance is blocked with a wooden stopper and glue, to simulate inundated conditions. A fiber-optic micro oxygen sensor is inserted through a hypodermic needle 2-5 mm into the nest cavity.

Fig. 10. O2 depression during and after a simulated inundation of the nest of Colobopsis anderseni. The arrows indicate the nest closing and opening. Data from Nielsen et al. (2009).

Fig.11. Oxygen concentrations in a closed respiratory chamber containing workers of Colobopsis anderseni. Data from Nielsen and Christian (2007).

Fig.12. Respiratory Quotient (RQ) for workers of Colobopsis anderseni in closed respiration chambers at different CO2 concentrations at the end of the experiment. Line (1) represents the CO2 concentrations with constant RQ, and Line (2) shows the CO2 concentrations associated with increasing RQ, representing increasing anaerobic respiration. Data from (Nielsen and Christian 2007)