insects Article Differential Gene Expression in Red Imported Fire Ant (Solenopsis invicta) (Hymenoptera: Formicidae) Larval and Pupal Stages Margaret L. Allen 1,* , Joshua H. Rhoades 2, Michael E. Sparks 2 and Michael J. Grodowitz 1 1 USDA-ARS Biological Control of Pests Research Unit, National Biological Control Laboratory, Stoneville, MS 38776, USA; [email protected] 2 USDA-ARS Invasive Insect Biocontrol and Behavior Laboratory, Beltsville, MD 20705, USA; [email protected] (J.H.R.); [email protected] (M.E.S.) * Correspondence: [email protected]; Tel.: +1-662-686-3647 Received: 16 October 2018; Accepted: 29 November 2018; Published: 5 December 2018 Abstract: Solenopsis invicta Buren is an invasive ant species that has been introduced to multiple continents. One such area, the southern United States, has a history of multiple control projects using chemical pesticides over varying ranges, often resulting in non-target effects across trophic levels. With the advent of next generation sequencing and RNAi technology, novel investigations and new control methods are possible. A robust genome-guided transcriptome assembly was used to investigate gene expression differences between S. invicta larvae and pupae. These life stages differ in many physiological processes; of special importance is the vital role of S. invicta larvae as the colonies’ “communal gut”. Differentially expressed transcripts were identified related to many important physiological processes, including digestion, development, cell regulation and hormone signaling. This dataset provides essential developmental knowledge that reveals the dramatic changes in gene expression associated with social insect life stage roles, and can be leveraged using RNAi to develop effective control methods. Keywords: fire ant brood; digestion; transcriptome; RNA-Seq; differential expression 1. Introduction Solenopsis invicta Buren (Hymenoptera: Formicidae), commonly referred to as the red imported fire ant (RIFA), is native to central South America and has been introduced to North and Central America, the Caribbean, Asia, Australia and New Zealand (http://www.cabi.org/isc/datasheet/50569, accessed 1 August 2017). One of the earliest introductions was to the Southern United States. First reported in Mobile, Alabama in 1930, invasive fire ants, including S. invicta, have since spread across most of the southern United States and may expand as far north as Virgina and west to Oregon [1,2]. Myriad ecological, agricultural and economic impacts have been reported [1,3,4]. In its native range, S. invicta is kept in check by predators, pathogens, and competition with other ant species, which are not present in the invaded areas. Development of safe and practical control mechanisms for S. invicta is important, both to protect native species and habitats, as well as to prevent economic impacts to agriculture and businesses. Several large-scale control attempts using the broad-scale insecticides Heptachlor, Dieldrin and Mirex were unsuccessful, resulting in extensive off-target effects impacting many species (reviewed in [1,5,6]). Ecologically friendly control methods are needed for S. invicta, which requires an intimate knowledge of important physiological processes. The S. invicta genome has been sequenced and is available in public databases [7]. Genomic and genetic data from ants have been used to study Insects 2018, 9, 185; doi:10.3390/insects9040185 www.mdpi.com/journal/insects Insects 2018, 9, 185 2 of 20 development [8,9], social evolution [10–12], social behavior [13–17], reproduction [18,19] and chemical communicationsInsects 2018, 9, x [20,21]. A microarray and cDNA library were deposited [8] for the express purpose2 of 21 of differential expression studies. While these data resources have been extraordinarily useful chemical communications [20,21]. A microarray and cDNA library were deposited [8] for the express for annotation [22] and broad comparisons relating to social form [19,23,24] and evolution [25], purpose of differential expression studies. While these data resources have been extraordinarily some studies have required and utilized further transcriptome sequencing. For example, differential useful for annotation [22] and broad comparisons relating to social form [19,23,24] and evolution [25], gene expression studies revealed 22 genes that differ between alate virgin and dealate mated queen some studies have required and utilized further transcriptome sequencing. For example, differential brains in S. invicta [26]. Because our study aim was to collect precise expressed sequences from the gene expression studies revealed 22 genes that differ between alate virgin and dealate mated queen samebrains colony in S. specimensinvicta [26]. thatBecause we intendedour study toaim use was for togene collect disruption, precise expressed we prepared sequences and from compared the transcriptomessame colony specimens from our targetthat we populations. intended to use for gene disruption, we prepared and compared transcriptomesSolenopsis invicta from isou anr target eusocial populations. insect; the ants live in large colonies within subterranean nests with interdependentSolenopsis invicta division is an eusocial of labor insect; among the individuals ants live in of large all life colonies stages andwithin multiple subterranean castes. Asnests with allwith holometabolous interdependent insects, division life begins of labor as anamong egg (Figureindividuals1A), whichof all hatcheslife stages and and then multiple feeds and castes. grows rapidlyAs with during all holometabolous the larval (Figure insects,1B) stage.life begins Once as large an egg enough, (Figure the 1A), larvae which void hatches gut contentsand then tofeeds enter pupationand grows as a pre-pupa,rapidly during then completethe larval a (Figure molt to become1B) stage. pupae. Once Thelarge pupa enough, is initially the larvae white (Figurevoid gut1C), andcontents darkens to (Figureenter pupation1D) as it as approaches a pre-pupa, the then adult comple moltte and a molt then to finally become emerges pupae. as The an pupa adult is (Figure initially1E). Ofwhite particular (Figure importance 1C), and darkens is the role(Figure of larvae1D) as init a providingpproaches thethe colonyadult molt with and a communalthen finally gut;emerges larvae haveas an been adult described (Figure 1E). as “aOf proteinparticular digestive importance organ is the for role the of colony” larvae in [27 providing]. Adult the ants colony lack digestivewith a proteasescommunal and gut; therefore larvae dohave not been digest described protein as efficiently “a protein [28 digestive,29]. Fire organ ant colonies for the primarilycolony” [27]. process Adult and digestants proteinslack digestive via late-stage proteases larvaeand therefore [30,31]. do Unique not di mouthpartsgest protein efficiently [32,33], a body[28,29]. shape Fire ant and colonies setae that formprimarily a food process basket [and1], salivarydigest proteins secretions via late-stage [28,34], and larvae filtration [30,31]. structures Unique mouthparts [35] make [32,33],S. invicta a bodylarvae uniquelyshape and capable setae ofthat digesting form a food complex basket molecules. [1], salivary Protein-rich secretions food [28,34], items and are filtration brought structures to the larvae [35] for make S. invicta larvae uniquely capable of digesting complex molecules. Protein-rich food items are digestion and subsequent regurgitation, a process called stomodeal trophallaxis [36]. While most adult brought to the larvae for digestion and subsequent regurgitation, a process called stomodeal workers do not have large protein demands, the queen requires an abundant, uninterrupted supply trophallaxis [36]. While most adult workers do not have large protein demands, the queen requires of protein for egg laying. The regurgitated food is collected by the nurse ants, who retain some but an abundant, uninterrupted supply of protein for egg laying. The regurgitated food is collected by feed the majority to the queen [37]. This “child labor” [36] leverages normal larval digestive processes, the nurse ants, who retain some but feed the majority to the queen [37]. This “child labor” [36] allowingleverages other normal members larval ofdigestive the colony processes, to forego allowing energy other and resourcemembers expenditures of the colony thatto forego are required energy to digestand protein-richresource expenditures food items. that The are changes required in to gene dige expressionst protein-rich associated food items. with The the juvenilechanges rolein gene of fire antsexpression have not associated been examined with the in juvenile detail. role of fire ants have not been examined in detail. FigureFigure 1. 1.Developmental Developmental stages stagesof of thethe S.S. invicta worker caste: caste: (A (A) )egg; egg; (B ()B larva;) larva; (C ()C early) early stage stage (white) (white) pupa;pupa; (D ()D late) late stage stage pupa pupa or or pharate pharate adult;adult; ((E) adult. RNA RNA samples samples were were ex extractedtracted from from representatives representatives similarsimilar to to (B (,BC,).C). GeneGene disruption disruption methods methods includingincluding RNA interference (RNAi) (RNAi) have have been been widely widely used used to tostudy study genes.genes. Systematic, Systematic, high high throughput throughput RNAiRNAi tools for identification identification of of pe pestst control control target target genes genes are are being being developeddeveloped