Insect Science (2019) 00, 1–12, DOI 10.1111/1744-7917.12727 ORIGINAL ARTICLE Ergatoid reproductives in the Neotropical termite Nasutitermes aquilinus (Holmgren) (Blattaria: Isoptera: Termitidae): developmental origin, fecundity, and genetics Iago Bueno da Silva1 , Ives Haifig2 , Edward L. Vargo3, Fabiana Elaine Casarin4, Marcelo Luiz da Mota5, Juliana Toledo Lima6 and Ana Maria Costa-Leonardo1 1Laboratorio´ de Cupins, Departamento de Biologia, Instituto de Biociencias,ˆ Universidade Estadual Paulista—UNESP, Rio Claro, SP, Brazil; 2Centro de Cienciasˆ Naturais e Humanas, Universidade Federal do ABC, Sao˜ Bernardo do Campo, SP, Brazil; 3Department of Entomology, Texas A&M University, College Station, Texas, USA; 4Departamento de Cienciasˆ Biologicas,´ Universidade Federal de Sao˜ Paulo—UNIFESP, Diadema, SP, Brazil; 5InstitutodeCienciasˆ Agrarias,´ Universidade Federal de Uberlandia—UFU,ˆ Monte Carmelo, MG, Brazil and 6Centro de Estudos em Recursos Naturais, Universidade Estadual de Mato Grosso do Sul, Dourados, MS, Brazil Abstract Termite colonies are usually headed by primary reproductives, which estab- lish nests during the swarming season. However, secondary reproductives may develop in some species and become supplementary or replacement breeders, extending colony lifes- pan. Here we investigate the developmental origin, fecundity and genetic characterization of ergatoid reproductives in the Neotropical termite Nasutitermes aquilinus (Holmgren), using morphometrical and histological techniques, five microsatellite loci and the COI mitochondrial DNA. Twelve measurements performed on 208 apterous individuals of N. aquilinus revealed 10 groups, including ergatoid females, which developed from major workers through two successive molts, and were characterized by the presence of imaginal features such as eyes and wing buds. The differentiation of these features was correlated to physogastric development in these ergatoids. Histology revealed oocytes in all matu- ration stages in worker-derived reproductives of N. aquilinus, presence of nonflagellate spermatozoa inside the spermatheca, and royal fat body. Thus, ergatoid reproductives were reproductively functional. According to the genotypes of 221 individuals from 11 nests, and mitochondrial haplotypes of 43 ergatoids, 73% of the colonies were simple families, whereas 27% were extended families. Despite the occurrence of related reproductives, low inbreeding rates were detected within and among colonies. Such values could be explained given that sib mating itself cannot result in a higher inbreeding rate but depend on several factors discussed in detail. This is the first study to investigate the genetic structure of termite colonies influenced by the development of ergatoids, and further investigations are encouraged to understand the influence of these reproductives on colony lifespan. Key words breeding system; caste differentiation; COI; genotyping; microsatellites; reproduction Introduction Correspondence: Ana Maria Costa-Leonardo, Laboratorio´ Termite colonies are usually headed by primary repro- de Cupins, Departamento de Biologia, Instituto de Biociencias,ˆ ductives, termed king and queen, which establish the new Universidade Estadual Paulista—UNESP,Rio Claro, SP 13.506- colonies after swarming (Roisin, 2000). This event may be 900, Brazil. Tel: +55 19 35264138; fax: +55 19 35264138; influenced by several factors and tend to be synchronized email: [email protected] among colonies, increasing the likelihood of unrelated 1 C 2019 Institute of Zoology, Chinese Academy of Sciences 2 I. B. da Silva et al. partners founding a new nest (Mitchell, 2008). In many The Neotropical termite Nasutitermes aquilinus (Holm- species, the breeding tasks of the colonies are performed gren) is an arboreal nest-building species classified as by secondary reproductives when the primary reproduc- an opportunistic pest in rural buildings (Torales, 1998; tives are absent. These individuals are neotenics when Fontes & Milano, 2002). Colonies of N. aquilinus are gen- differentiated from immatures or adultoids when differ- erally headed by a founding pair but, according to Torales entiated from winged sexuals (Myles, 1999). Neotenic and Coronel (2006), about 20% of the colonies develop reproductives develop from workers, referred to as erga- ergatoid reproductives during their lifecycle. Ergatoid toids, or from nymphs, referred to as nymphoids (Roisin, reproductives may be numerous when present, occurring 2000), and their occurrence is less prominent within the in dozens or even hundreds of individuals, but only their so-called higher termites, in which these individuals are external morphology has been examined in previous re- replaced by adultoid reproductives in some Termitidae search studies (Fontes & Terra, 1981; Torales & Coronel, lineages (Myles, 1999). 2006). Thus, this study aimed to explore the developmen- Sexual reproduction is predominant within Isoptera, tal origin, fecundity and genetics of the ergatoid reproduc- resulting in a higher genetic diversity which has a tives of N. aquilinus, trying to answer the following funda- positive impact on colony survival during environmental mental questions: How do ergatoid females differentiate stress. Some species conditionally alternate from sexual within the apterous line? Are those individuals functional to asexual reproduction, solving this breeding tradeoff reproductives? How are the colonies of N. aquilinus (Matsuura, 2011). Thus, while the genetic diversity is genetically structured? To answer these questions, we maintained through sexual reproduction, the queen may used different methodological approaches, including adopt asexual reproduction to duplicate her genetic morphometry, histology, and genetic analyses using mi- contribution and increase within-colony relatedness crosatellite markers and mitochondrial DNA sequencing. (Matsuura et al., 2009). In both sexual and asexual reproduction, the secondary reproductives may extend Materials and methods the colony lifespan (Vargo, 2019). Termite colonies may be classified into the following Sampling of termites breeding structures: simple families, in which a single pair of monogamous reproductives and its progeny are present; A total of 12 nests (#1 – #12) of N. aquilinus (Holmgren, extended families, in which multiple related reproductives 1910) were collected from urban and agricultural areas descend from a royal couple and perform the reproduc- in the cities of Rio Claro (22°2443.9S47°3634.0W), tive tasks; and mixed families, characterized by the co- Sao˜ Carlos (21°5926.7S47°5258.3W), Corum- existence of unrelated reproductives and their progeny bata´ı(22°1326.2S47°3826.1W), and Itapetininga (Vargo & Husseneder, 2011; Vargo, 2019). In any case, (23°354.1S; 48°159W), State of Sao˜ Paulo, Brazil many factors are responsible for the genetic structure of (Fig.1,Table1). the colonies, including number and relatedness among primary and secondary reproductives, and the number of descendant generations (Thorne et al., 1999; Vargo & Morphometry Husseneder, 2011). The genus Nasutitermes is the most diverse in num- Sampled termites from #1, #2, and #12 were fixed in ber of species within Isoptera and show plentiful variety FAA(formaldehyde, absolute alcohol, and glacial acetic of breeding mechanisms. The occurrence of multiple sec- acid, 1 : 3 : 1) during 24 h and transferred to 80% ethanol. ondary reproductives is common, which exhibit an impor- All the samples were analyzed under stereomicroscope tant role in egg-laying rate and contribute to the population Zeiss Stemi SV11 (Zeiss, Jena, Germany) and 12 different growth (Roisin & Pasteels, 1986, 1987; Lefeuve, 1987; measures were obtained following the methods described Noirot & Thorne, 1988). Few studies have focused on the by Haifig and Costa-Leonardo (2016). In total, 208 genetic structure within Nasutitermes, shedding light on apterous individuals were measured: 45 larvae, 30 minor the origins and relatedness of the reproductives, as well workers, 30 major workers, 15 minor soldiers, 45 major as the inbreeding rate in colonies headed by them (Atkin- soldiers, 30 presoldiers, 3 preneotenics, and 10 ergatoid son & Adams, 1997; Thompson & Hebert, 1998; Adams females. The measures used were: (1) head length (HL), et al., 2007). However, no genetic investigation has fo- including the nasus in the case of soldiers; (2) maximum cused on the ergatoid reproductives, which differentiate head width (HW1); (3) head width at the base of the in about 36% of the Nasutitermes spp. studied up until mandibles (HW2); (4) maximum head height without now (Silva et al., 2018, unpublished data). postmentum (HH); (5) labrum width (LW); (6) pronotum C 2019 Institute of Zoology, Chinese Academy of Sciences, 00, 1–12 Ergatoid reproductives in Nasutitermes aquilinus 3 Fig. 1 (A) Arboreal nest of Nasutitermes aquilinus. (B) Ergatoid females (ef) and major workers (Mw). length (PL); (7) pronotum width (PW); (8) mesonotum based on the cylinder shape. To estimate a correlation be- length (MsL); (9) metanotum length (MtL); (10) tibia tween physogastry and development of imaginal features, length (TL); (11) femur length (FL); and (12) femur width the right anterior wing bud length was also measured, and (FW). Morphometric data were submitted to a principal the data were subjected to the Pearson’scorrelation coeffi- component analysis (PCA). The scores of PC1 and PC2 cient. All statistical tests were performed using R version were analyzed using ANOVA (one-way),