Molecular Ecology Notes (2003) 3, 355 – 357 doi: 10.1046/j.1471-8286.2003.00448.x © 2003 Blackwell Publishing Ltd Blackwell Publishing Ltd. PRIMER NOTE Isolation and characterization of six microsatellite loci in the drywood termite Cryptotermes secundus (Kalotermitidae) A. FUCHS,* J. HEINZE,* C. REBER-FUNK† and J. KORB* *Department of Biology I, University of Regensburg, Universitätsstraße 31, D-93040, Regensburg, Germany, †ETH Zürich, Ecology & Evolution, ETH-Zentrum NW, CH-8092 Zürich, Switzerland Abstract The Kalotermitidae is a family of lower termites and its species are ideal to study the evo- lution and maintenance of eusociality within the Isoptera. Co-dominant genetic markers are an essential tool to elucidate the putatively complex colony and population structure. Six polymorphic microsatellite loci were identified in the drywood termite Cryptotermes secundus (Kalotermitidae). To test the variability of the markers, 18 colonies (n = 18) from Darwin (Australia) were assayed. We found two to eight alleles per locus with the level of observed heterozygosity at each locus ranging from 0.00 to 0.53. The significantly positive F IT value suggests that some kind of inbreeding does occur in this population. Keywords: Cryptotermes secundus, Kalotermitidae, microsatellites, termites Received 2 February 2003; revision accepted 17 March 2003 In recent years, microsatellite genetic markers have been developed for several species of Isoptera. However, the focus was mainly on the higher termites (Termitidae) (Thompson et al . 2000; Vargo & Henderson 2000; Vargo 2000; Hayashi et al . 2002) and, among the lower termites, the Rhinotermitidae (Harry et al . 2001; Kaib et al . 2000). For the lower termite family Kalotermitidae, no genetic markers are available so far. In Cryptotermes secundus (Kalotermitidae) (Hill 1942), both monogamous and polygamous colonies can be found and, upon the loss of one or several primary reproductives, replacement reproductives can emerge from helper or nymphal stages (J. Korb, unpublished studies). As a consequence, inbreeding occurs between primary and replacement reproductives and also among replacement reproductives. This might result in a complex genetic structure, both at the colony and the population level. As allozyme analysis failed to detect any polymorphisms ( J. Korb, unpublished studies), we constructed a DNA library and searched for microsatellite genetic markers following the protocol of Tenzer et al . (1999) and Gautschi et al . (2000). Here we characterize six polymorphic microsatel- lite loci for C. secundus . Heads of nonreproductives from a mangrove area near Palmerston-Channel Island in Darwin Harbour (Northern Territory, Australia; 12 ° 30 ′ S, 131 ° 0 ′ E) were ground in liquid nitrogen. DNA was extracted from several individuals from three (for loci Csec 1–3) or six (for loci Csec 4–6) colonies, respectively following a modified cetyltrimethyl ammonium bromide protocol (Sambrook et al . 1989). Approximately 1 μ g DNA was digested with Tsp 509 I, resulting in fragments of mainly 200 –1000 bp. For loci Csec 1–3 the Tsp 509 I-digested DNA was size selected for 200 – 1000 bp fragments using DEAE membranes (Schleicher and Schuell). Two adaptors (Tsp AD short and Tsp AD long; Tenzer et al . 1999) were ligated to the DNA, result- ing in blunt-ended fragments. After purification with Ultrafree-4 spinning columns (Millipore), the ligation prod- uct was amplified using one of the adaptors (Tsp AD short) as a primer. Polymerase chain reaction (PCR) was carried out in a final volume of 25 μ L containing 1 μ L ligation product, 0.25 μ L Taq DNA polymerase (5 U/ μ L; Pharmacia) and a final concentration of 1 μ m Tsp AD short, 1 × buffer (with 1.5 m m MgCl 2 ; Pharmacia) and 250 μm of each dNTP. We used a Biometra T1 thermocycler (Whatman) (which was also used for all subsequent PCRs) and the following thermo-treatment was applied: initially 72 ° C for 5 min to synthesize the nick between the linker and the genomic DNA, followed by 30 cycles of initial denaturation at 93 ° C for 1 min, primer annealing at 55 ° C for 1 min and Correspondence: A. Fuchs. Fax: 49 941 943 3304; E-mail: alexander.fuchs@biologie.uni-regensburg.de