Molecular genetics is “a branch of genetics dealing with the structure and activity of genetic material at the molecular level.” Essentially, it is the application of various molecular techniques, for example, polymerase chain reaction (PCR) and gel electrophoresis , to investigate aspects of an organism’s genetic material. These molecular techniques have many practical applications, from forensic investigations to conservation of endangered species to termite research.
Termites are economically important insect pests that have an interesting and complicated biology. Investigation of termite biology is challenging due to the cryptic nature of the termite nest that is difficult to sample entirely. Also, individual termite species are difficult to distinguish since most morphological identification methods require alates or soldiers, further complicating studies of their biology. To overcome some of these difficulties, molecular genetic techniques have been utilized to further investigate termite biology. Applications of molecular genetics in termite research include clarification of systematics and taxonomy, understanding caste differentiation, identification of species and invasive species surveillance, discovering relationships among populations and unraveling family structure within a colony (see review by Vargo and Husseneder 2009).
For this article, we will focus on two examples of genetic research for termites involving species identification and relationships among populations.
Case study 1:
Formosan subterranean termite molecular diagnostics technique
The Formosan subterranean termite (FST), Coptotermes formosanus, is an invasive subterranean termite that was introduced into the United States in the 1950s from southern China. Since its introduction, it has spread throughout the southeastern U.S. and Texas (Evans et al. 2013). This termite is particularly damaging since it can feed on the heartwood of living trees, rather than solely on dead wood (Lai et al. 1983). Annually, Americans spend more than $1 billion in treatment and damage from the FST (Lax and Osbrink 2003, Raina 2004).
Arguably, the first step to controlling the FST is identification. Dichotomous keys have been developed for identification of soldiers and alates (Scheffrahn and Su 1994). These keys do not work on workers, and therefore, cannot be used on small samples that do not contain the necessary castes for identification. DNA sequence data have been used to identify FST from other subterranean termites (Smith et al. 2010) but this method is expensive and time-consuming.
Szalanski et al. (2004) developed a molecular diagnostic technique, but this method requires two polymerase chain reactions (PCRs) (see “PCR” article, page 58), which doubles the chances of error in the PCR process. The objective of our research was to develop a multiplex PCR protocol to identify FST from other termites using only a single PCR reaction.
Termites were used from a variety of locations and species (see Table 1, right), and identified using the keys of Scheffrahn and Su (1994). DNA was extracted and used to develop universal primers. The universal primer set was developed to serve as a positive control to distinguish between an unsuccessful PCR and a negative result. A FST specific primer (from Szalanski et al. 2004) was also used in the multiplex PCR to differentiate the FST from all other termites. In the multiplex PCR, one fragment of DNA is amplified, or copied, in all samples while a second DNA fragment is amplified in only the FST (see Figure 2, below). To test its efficacy, this method was PCR screened on 69 DNA samples and the PCR primers screened on 1,304 mtDNA 16S DNA sequences samples using Geneious software (see Table 1, right).
Overall, the new genetic diagnostic method for differentiating the FST from other termites was successful. This method is simpler and faster than previous methods (Szalanski et al. 2004, Evans et al. 2013). Our method also allows for identification of any life stage of termite, which will be useful in monitoring the spread of the FST throughout the United States and minimizing its damage. Now, any sample sent in for identification can successfully, and quickly (as little as four hours), be determined. (For more details, see Janowiecki and Szalanski 2015).
Case study 2:
Incisitermes schwarzi population genetics
Drywood termites (family Kalotermitidae) are a group of termites consisting of 21 genera containing 456 species. In the United States, there are four species of Incisitermes, two species of Cryptotermes and one species of Marginitermes (Krishna et al. 2013). Drywood termite biology differs from subterranean termite biology in several main aspects. Drywood termites have no worker caste to maintain the colony. Instead, they rely on pseudergates, which are more fluid as to what caste they may become. Also, drywood termite nests are smaller, consisting of up to 2,500 members, and these nests do not connect to the soil as subterranean termite nests do. In the United States, only Incisitermes minor and Marginitermes hubbardi are economically significant with the annual cost of damage and treatment of I. minor alone costing $250 million (Cabrera and Scheffrahn 2001).
Incisitermes schwarzi is a drywood termite that was described in 1919 by Nathan Banks (Banks 1920). This species is found from southern Florida in the United States to northern South America. The objective of this study was to characterize the genetic diversity of I. schwarzi from geographical locations across its range.
Seventeen termite samples were collected from a broad geographical range. Portions of the 16S and COII region of mitochondrial DNA was sequenced and aligned with Geneious software. For the 16S region sequences, there was 0.3 to 9.1% genetic variation. This means that out of the about 440 base pairs in the sequence, the most divergent sequences contained 9.1 percent of different base pairs (or about 40 different base pairs). For the COII region, there was slightly more genetic variation ranging from 0.6 to 10.0 percent. When assigning haplotypes, which are sequences that vary by at least one base pair, the 16S haplotypes were all unique except two samples from Florida and two samples from Barbados. This means two unique samples from Florida had identical sequences, as did the two unique samples from Barbados. All other samples varied by at least one base pair. For the COII haplotypes, all haplotypes were unique, meaning that no two COII sequences were identical. The 16S and COII sequences were combined to create a phylogenetic tree (see Figure 1, above). Our results are similar to a similar previous study by Austin et al. (2012) (see Table 2, below). Generally, our wider sampling range likely caused more single sample haplotypes and a higher 16S genetic variation than was seen in Austin et al. (2012).
The preceding two examples of molecular genetic techniques are a small sampling of the potential that these research techniques have for investigating termite biology. Coupling these methods with other traditional research methods, many of the cryptic aspects of termite biology have the potential to be examined.
Relating these molecular techniques to practical applications, termite genetics can be an important aspect in control of pest species. The first case study, developing a molecular technique to differentiate the Formosan subterranean termite from other termites, could be used to help identify if a problematic termite infestation is a native species or the invasive FST. Because of the different biological aspects of the FST (i.e., larger colonies, attacking life trees, etc.), identification may be critical before successful control can occur.
Regarding the second case study, while it may not seem as directly practical, this technique of determining relatedness of populations can be useful. For example, these population genetics techniques can be used in determining the origin of an invasive species or a new pest population. By determining a population’s nearest genetic relative, aspects of its transport and spread may be inferred. This can lead to a better understanding of the pest that could help slow or stop its spread.
While these genetic methods will not directly kill termites, their benefits will greatly assist in management of pest termite species. And although these techniques currently are expensive and difficult, the future of this technology will likely incorporate larger data and become much cheaper and accessible. Ideally, if this technology becomes even more accessible, it may become a more integral component to control these pests.
What is ‘PCR’?
The polymerase chain reaction (PCR) is a method of replicating specific portions of an organism’s genetic material.
Developed in 1983 by Kary Mullis, this method greatly simplified previous replication techniques, allowing for a rapid growth in genetic research. PCR uses repeated heating and cooling cycles to replicate the DNA and is performed in a specialized piece of equipment called a thermal cycler (pictured, right). The DNA is heated to approximately 94-98°C to denature the DNA. This denaturing splits the double helix apart, allowing specific primers to bind. Primers are small base pair strings (about 20 bp) that are designed to be specific to a DNA region flanking the target region. These primers bind to the denatured DNA during the annealing step where the temperature is lowered to 50-65°C. The replication occurs during the extension step where the temperature is held at 75-80°C. A heat tolerant Taq polymerase is used to place individual base pairs onto the new DNA fragment since it is able to survive the denaturing step. This denaturing-annealing-extending cycle is repeated 35-40 times, thus yielding millions of copies of the selected region of DNA. This large number of copies can then be used to sequence the fragment of interest.
The authors are with the department of entomology, University of Arkansas, Fayetteville, Ark.
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