Many species of social Hymenoptera demonstrate behavioral flexibility, where older workers that typically forage can revert to younger worker tasks, such as nursing, when these are absent. This flexibility is typical of the sterile worker class, yet rare in queens. In the little fire ant (Wasmannia auropunctata) queens have been reported to perform only egg laying. We examined behavior of queens of W. auropunctata after demographic manipulation. When half of the workers were removed from the colony, queens were observed caring for eggs, larvae and pupae as well as eating outside of the nest, like forager workers. We examined the relationship between these atypical queen behaviors and their juvenile hormone binding protein (JHbp) and vitellogenin (Vg) expression via QRT-PCR method. JHbp and Vg expression decreased when queens were performing worker tasks, resembling the expected expression pattern of typical sterile workers. Flexibility in queen behaviors in the little fire ant may be an important adaptation to changing environments. As a significant invasive species, such adaptation may increase the probability of colony survival during propagation. Our results not only present new insights in behavioral flexibility in social insects, but also increases our understanding of the success of this significant invasive species.  

Samples

Nests (N=20) of the little fire ant W. auropunctata were collected from dry twigs and leaf litter in the northern region of the Caribbean island of Puerto Rico, and identified by morphology. They were housed in artificial nests which consisted of plastic boxes (25cm x 13cm x 7.5cm) coated with Fluon (Northern Products Inc., Alsip, IL.) on the sides. The boxes’ lids were perforated with a pin to allow air exchange. The nesting area within the nest box consisted of a 3cm² piece of thin (less than 1cm thick) wood elevated 0.5cm by a strip of clay placed along the edges of the wood.  Each nest was kept at 25°C, a relative humidity (RH) between 80% and 85%, and 12 hour light cycles. Nests were fed daily with 0.5g of feeding mixture (Hölldobler & Wilson 1994) placed in a feeding arena within the box at approximately 10cm from the nesting area. Nests used in the study had multiple queens, eggs, larvae and pupae. Queens which can be distinguished from the workers by their size (workers are 1.2-1.5mm, while queens are 4.5-5mm; Wetterer & Porter 2003) were identified by morphology (n=52     ) and painted on the thorax and/or abdomen with one color or a combination of colors with nail polish.

Behavioral flexibility of queens assays

Six nests (N=6) with a total of 19 queens were used for behavioral flexibility assays. Colonies were kept in the artificial nests three days prior to the start of experiment observations and fed daily. During the experiment observation period, queens were observed for 10 min, daily in a total of nineteen days; food was removed after each observation period. The 10min observation period was determined after observing that the proportion of behaviors does not vary significantly in 10, 15, 20 and 30 minute intervals. Observations were made for different tasks, including egg laying, a typical behavior, and nursing (i.e. manipulating brood) and foraging (i.e. queens seen feeding in the designated arena) which are non-typical behaviors. Behaviors were tabulated by number of events e.g. number of eggs laid, number of times brood was manipulated and number of times queens walked to feeding arena and was seen feeding, during the observation periods. Control observation period was performed for a period of five days. After the first five days of observations, worker population per nest was estimated via nest pictures and approximately 50% of workers were randomly removed, including both nurses and foragers. This percentage of worker removal is based on related research on worker division of labor and plasticity, where each worker caste constitutes to about the 50% of the worker force (Rivera-Marchand & Fernández-Casas unpublished) and to resemble loss of workers in nature. Observations for the experimental period, continued for fourteen days and queen behaviors were classified and noted (Table 1     ) as explained prior. The remaining nests were used to measure JHbp and Vg gene expression.

Bioinformatic analysis and primer design

Primers were designed for gene sequences related to JH and Vg expression. Since JH is a      terpenoid its gene expression levels were determined indirectly by measuring Juvenile hormone binding protein (JHbp), an associated protein. JHbp is directly correlated with the onset of JH production in the hemolymph (Kramer et al. 1976; Shemshedini & Wilson 1990) since it prevents the absorption and enzymatic hydrolysis of JH, and maintains a steady reservoir of the hormone in the hemolymph; hence free JH is virtually absent (Roe &Venkatesh 1990; Tan 2007). Also, JHbp/JH interaction is specific and of high affinity (KD=10^-9M), more than 99% of JH is bound to JHbp (De Kort & Granger 1996; Tan 2007). Other studies have further suggested this direct involvement as well (Prestwich et al. 1996; Hagai et al. 2007     Sequences for JHbp and Vg of W. auropunctata were obtained from NCBI Gene Bank. Vg sequences (XM_011697672.1, XM_011697673.1) were aligned using MAFFT (Multiple sequence alignment tool: Katoh et al. 2009). Primers (Table 2) were designed using primer3 from NCBI (Ye et al. 2012) with the obtained consensus sequence for Vg and the JHbp sequence (XM_011708554). Actin and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) were used as housekeeping genes (Wong & Medrano 2005; Scharlaken et al. 2008).

RNA extraction, cDNA and qPCR of JHbp and Vg.

Fourteen nests (N=14), different from the ones used in the first behavioral assay, were used with a total of 33 queens. Nests were randomly assigned to control or experimental groups (nest with workers removed), behavioral assays were repeated. Seven days after worker removal, queens were collected by tasks; from control nests n=15 and from experimental nests n=18 and placed in a microtube with 20μl of RNAlater reagent (Qiagen Valencia, CA) stored at -80°C. RNA extraction was performed using the RNeasy Mini Kit (Qiagen). RNA was quantified for each sample using a Nanophotometer (Implen, Westlake Village, CA), where the minimum amount of RNA was 10μg/μl. RNA was normalized and treated with DNase 1, following a protocol developed by BioLabs (Ipswich, MA.). cDNA was synthesized using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad Hercules, CA.) following the manufacturer's protocol with 10µl of RNA as a template. cDNA synthesis was verified in an electrophoresis 1% ETBR-gel.

qPCR was performed using the MJ Mini-Opticon Real-Time PCR (Bio-Rad) with a two-step amplification program with post-amplification melt curve analysis. As a standard for quantification purposes, actin and GAPDH were used as reference genes (Wong & Medrano 2005; Scharlaken et al. 2008). Reactions were prepared with 2µl of first strand cDNA as a template in a master mix of 1µl of each forward and reverse primers per gene at 10nM and 5µl of iTaq Universal SYBR Green Supermix (BioRad) in a final volume of 10µl. Relative gene expression was calculated using the geometric mean analysis method (Vandesompele et al. 2002), using the following equation:

Relative gene expression=(E_GOI)^∆CtGOIGeoMean[(E_REF)^∆CtREF],

where E= primer efficiency, GOI= gene of interest, GeoMean= geometric mean and REF= reference gene. ΔCt was calculated using the average Ct values of the control group for each gene (calibrator Ct). The relative expression values presented are relative to the control group.