Survival of Coptotermes testaceus ( Isoptera : Rhinotermitidae ) to Environmental Conditions ( Relative Humidity and Temperature ) and Preference to Different Substrates

Coptotermes Wasmann (Isoptera: Rhinotermitidae), is a genus of subterranean termites that currently has 21 validated species (Chouvenc et al., 2016), of which 16 are classified as pests of economic importance in various parts of the world (Krishna et al., 2013). While only two of these species, C. formosanus Shiraki, and C. gestroi (Wasmann) have demonstrated remarkable invasive abilities, most Coptotermes species still have an economic impact in their native range. Coptotermes have the ability to infest live trees, create large, Abstract Coptotermes testaceus (L.) (Rhinotermitidae) is a subterranean termite species that causes damage in urban and agricultural areas in the neotropics. Despite its economic importance, there are no studies on its basic biological aspects for laboratory management and the development of strategies for its control. The objective of the present study was to evaluate the relative humidity, temperature, substrate moisture and preference to different wood substrates for the best C. testaceus survival under laboratory conditions. For this, a range of eight relative humidity (from 9 to 100%), three temperatures (20, 25 and 30 °C), six substrates (Pinus sp, Cedrela odorata (L.), Cocos nucifera (L.), Eucalyptus urophylla (S. T. Blake), Haematoxylum campechianum (L.) and Tabebuia rosea [Bertol.] DC) and four substrate moistures, (0 to 60%) were tested. The results of this study indicated a significant effect of all factors on termite survival or termite preference. When tested independently, the highest survival percentage of C. testaceus was obtained with humidity of 100%, temperature of 20 °C, substrate moisture of 60% and the Eucalyptus urophylla substrate, reaching 83.33% survival at 21 days of observation. With these preliminary assays on small termite groups, it is concluded that with the appropriate percentages of humidity, temperature and substrate and the interaction of these three factors, further research can be conducted using larger termite groups in biologically relevant conditions, in order to study various aspects of C. testaceus biology. Sociobiology An international journal on social insects

Due to the economic importance of C. testaceus as a pest, it is necessary to find strategies for its control. However, to date, the environmental requirements to keep populations alive for prolonged periods of C. testaceus in the laboratory that allow the establishment of bioassays to evaluate their control, have not been reported. On the other hand, there is a continuous search for friendly alternatives to the environment and human health, for termite control. These studies generally start with laboratory bioassays, for example, the use of target-specific chemicals (baits and termiticides). While a lot of research efforts have been focusing on botanicals (essential oil, seed, bark, leaf, fruit, root, wood, resin) and entomopathogens (fungi, bacteria and nematodes), none has resulted in practical or commercial application (Verma et al., 2009;Chouvenc et al., 2011).
In order to provide preliminary information of the basic biological requirements for the survival and maintenance of C. testaceus in laboratory conditions, the aim of this study was to evaluate the survival of C. testaceus at various levels of relative humidity, temperature, substrate moisture, and preference for different substrates, to obtain the appropriate conditions that allow establishing live termite colonies in the laboratory, to ultimately carry out bioassays to study the control of C. testaceus.

Obtaining C. testaceus
The specimens were collected in the botanical garden of the Academic Division of Biological Sciences of the Universidad Juárez Autónoma de Tabasco. Traps (modified from Tamashiro et al., 1973) built with a metal cylinder (18.5 cm high x 15.5 cm in diameter) with exposed ends and internal walls lined with wood were used, a roll of corrugated paper 10 cm in diameter was placed in the center. The trap was placed in the basal part of trees that had damage caused by C. testaceus, buried in the ground 20 cm deep with the top of the trap covered. The corrugated paper cylinder with termites was removed after five days and transferred to the laboratory. In this study only workers were used of a single colony (a total of 1740).

C. testaceus survival at different percentages of relative humidity (RH) and temperatures
The methodology for this section was proposed by Zukowski and Su (2017), with modifications. Environmental chambers (EC) were conditioned using plastic containers with lid (24.7 × 17 × 6.4 cm), with a 2.7 cm diameter hole in the central part of the lid to introduce a digital hydrometer. The RH inside the EC was stabilized using various stabilizing materials (RHSM) in different amounts (Table 1). To achieve a high RH, the water was placed in plastic containers (6 cm diameter x 3.6 cm height) and cotton, covering the bottom of the EC; the salts and the silica gel were placed in the plastic containers described above and for the lowest RH, the CaCl 2 was spread at the bottom of the EC. The RH in the EC was assessed for 15 days with a digital hygrometer (VWR; TRACEABLE ™). The temperature was stabilized at 20, 25 and 30 °C in an incubator (NOVATECH, MOD. . The bioassay was carried out using five Petri dishes in each stabilized EC, the Petri dishes (60 mm x 15 mm) contained a filter paper disk (60 mm) to facilitate locomotion of the termites and as a source of food was pine sawdust (200 mg), 10 termite workers were placed in each box. The daily record of live termites was monitored, reporting the survival percentage. Dead termites were removed daily. The bioassay required five repetitions with ten workers in each repetition for the eight RH at the three temperatures tested (a total of 1,200 workers).
A device was designed to allow a high RH and for termites to choose between different substrates (Fig 1). Small environmental chambers (sEC) were used, which consisted of 8 x 5.5 cm (diameter x height) circular plastic containers, which inside contained plastic vials of 4 x 2 cm, suspended inside each sEC. Cotton was placed at the bottom of each radial sEC with water to obtain the RH of 100% and the substrates were placed in the vials (Fig 1a). Using 6 x 1 cm plastic tubes, six radial sEC were connected to a central chamber. The device was placed in the incubator at 20 ºC and the RH and temperature were stabilized for 4 h. Once the RH and temperature had stabilized, 30 termites were deposited in the central chamber and at 24 h the number of termites within each sEC with substrate was recorded, the termites found in the connection tubes were also included in the counts. The aggregation of termites to substrates was considered as preference. The RH and temperature were monitored during the bioassay and the percentage of preference was reported. The bioassay required ten repetitions with 30 workers in each repetition (a total of 300 workers).

Survival of C. testaceus at different substrate moisture
The most preferred substrate previously conditioned was used. In Petri dishes (60 mm x 15 mm) 3 g of the substrate were placed and sterile distilled water was used to obtain substrate moistures (SH) of 0, 20, 40 and 60%.The formula used to obtain the desired SH (NMX-AA-16-1984) was as follows: H = G -G1 x 100 G Where: H = % of humidity G = Wet sample weight in g G1 = Dry sample weight in g Six Petri dishes were placed in environmental chambers (used in the survival test at different RH and temperature) with the stabilized RH of 100 ± 0.25% (Fig 2a). Groups of ten termites were placed in each Petri dish with the substrate at the required humidity (Fig 2b). This bioassay was incubated at 20 °C in total darkness. The daily count of living termites was recorded, and the survival (percentage of live termites) was reported. The bioassay required six repetitions with ten workers in each repetition for the four SH evaluated (in total 240 workers).

Experimental design and statistical analysis
For all bioassays, a completely randomized simple design was used. The statistical analysis used for the termite survival test at different RHs and temperatures was an analysis of variance (ANOVA) for an 8 by 3 factorial experimental design (8 RHs and 3 temperatures), the response variable was the percentage of survival. Statistical analysis for preference towards different substrates was performed with a simple analysis of variance (ANOVA) and the percentage of preference as the response variable. A comparison of means with Fisher's LSD with an α = 0.05 was performed for these two trials. Finally, for the survival of termites towards different SH, a Kaplan-Meier LogRank analysis and the comparison of Holm Sidak means with an α = 0.05 were used, where the highest median lethal time (LT 50 ) was considered to select the best treatment. Because the results of the first two trials were expressed as a percentage, it was necessary to transform the data to the square root of the arcsine prior to ANOVA. The statistical package used for all analyzes was SigmaPlot 12.0. Table 2 shows the survival rate of C. testaceus 24 hours after being exposed to eight RH and three temperatures. The RH of 100% presents the highest percentage of survival at 24 hours of observation for the three temperatures evaluated (F = 109,313; df = 7; P <0.001). There was a greater survival of termites at a temperature of 20 °C (F = 192,213; df = 2; P < 0.001).

C. testaceus survival at various RH levels and temperatures
On the other hand, the interaction between the two studied factors, in the same way, generated a significant effect (F = 7,885; df = 14; p < 0.001), being the combination of 20 °C and 100% RH the treatment that provided the highest termite survival, with 88.4 ± 0.07%. Likewise, it was observed that as the RH decreases, the termite survival also does it gradually, while at higher temperatures, it's a lower survival.

C. testaceus preference for various substrates (woods)
Termites showed a statistically different response in their aggregation toward a substrate after 24 hours of observation (F = 4,630; df = 5; p < 0.001, Fisher's LSD α = 0.05). The most preferentially aggregated on eucalyptus, with 68.75%, followed by blackwood with 12.5% and pine with 10%. The substrates that obtained the lowest preferences were coconut fiber, pink poui and cedar, with 4.38, 4.37 and 0.0% respectively (Fig 3).

Discussion
The results of this study showed that the highest survival (88.4%) of C. testaceus was at 100% RH and temperature of 20 °C. This shows that both RH and temperature are important for the survival of C. testaceus, but they are even more so when the interaction between these factors occurs. This agrees with that reported by Gautam and Henderson (2011) as well as Wiltz (2012), who found the highest survival percentages for C. formosanus at low temperatures (20 and 10 °C) and high RH (98 and 99%) respectively. The results of this study show an effect of RH and temperature, however, these two factors alone cannot maintain the in vitro survival for long periods of time (24 h for this study), which is necessary for studies focusing on control aspects of this pest. Lenz (2005) points to substrate as another factor that must be considered to obtain successful results in bioassays. The substrate can be used for the construction of their galleries, food or as indicated by Hu et al. (2012) and Zukowski and Su (2017), a resource for obtaining moisture. That is why, in this study, the preference that C. testaceus had towards six different woods that could function as a substrate was also evaluated. The results obtained in this work indicated that eucalyptus was the substrate with the greatest choice. This coincides with studies carried out in agricultural systems where it was reported that C. testaceus was the main responsible for infestations in eucalyptus plantations (Amaral et al., 2004). This could be due to the fact that, as Zabel and Morrel (1992) points out, the different kinds of wood have the presence of extractable (chemicals). The part of the wood in which the extractables are produced is what determines if they play a role as attractants or repellents, i.e. in the sapwood, there is a higher concentration of starch and carbohydrates which makes this part of the wood more palatable to the biological agents that attack it. On the other hand, the heartwood has a series of components that make it less preferred to these agents (Kollmann, 1959). This leads us to think that eucalyptus was the most attractive substrate due to its chemical composition, in addition to cellulose, which is the main food source of xylophagous insects, such as termites (Bignell & Eggleton, 2000;Ramírez & Lanfranco, 2001;Shimada & Maekawa, 2010). This coincides with that reported by Scheffrahn (1991), who likewise attributes the preference of termites to this type of (extractable) substances.
Finally, and once the most preferred substrate was identified, different humidity levels were evaluated in this substrate. McManamy et al. (2008) pointed out that the humidity of the substrate is an important factor for the prolonged survival of species of subterranean termites, this due to the fact that after subjecting Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) to ideal conditions of temperature and RH, but exposed to low moisture wood, Percentage of survival of Coptotermes testaceus workers after being exposed to four substrate moistures (0%, 20%, 40% and 60%) at 20 °C and 100% RH, n = 6. Different letters on substrate moisture indicate significant differences (Holm Sidak, α = 0.05).
the termites did not survive. Our results indicated that the humidity of the substrate of 60% (the highest tested), was the one with which the longest survival time of the termite (21 days) was reached, reaching 83.33% of live insects at the end of this time. This coincides with that reported by Zukowski and Su (2017) for the termite species C. formosanus, which, after three weeks of observation (21 days), reached 90% survival with wet food, but when the food source provided was dry, survival was 0%. These results lead us to agree with what was pointed out by Gautam and Henderson (2014), who pointed out that subterranean termite species are extremely susceptible to desiccation, therefore, they require not only high RH, but also other sources of humidity for its greatest survival. At a global level, although studies of this type were conducted on pest species of subterranean termites (Rhinotermitidae), the current study provides preliminary information on optimal experimental and rearing conditions in the laboratory for C. testaceus. As previously shown, the importance of the biological relevancy of a bioassay when testing control method against subterranean termites in laboratory conditions is critical (Su, 2005;Chouvenc, 2018), to study the applicability of such approach in a field situation. The current study therefore provides initial guidelines for the manipulation of C. testaceus in the laboratory, thus being able to carry out future studies aimed at testing control approach for this species.

Acknowledgments
Cordial thanks to the editor and referees for their reviews and comments on our manuscript. To the Universidad Juárez Autónoma de Tabasco, for facilitating access to the laboratories, materials, and equipment of the Academic Division of Biological Sciences (DacBiol), the Academic Division of Agricultural Sciences (DACA), the Academic Division of Multidisciplinary of Jalpa de Mendez (DAMJM).To the National Council of Science and Technology (CONACYT) of Mexico for the scholarship awarded to carry out this study.

Contribution of the authors
CO Pozo-Santiago, conception, design, data collection, analysis, interpretation of results and document writing; M Pérez-De la Cruz, conception, design, critical review for important intellectual content, interpretation of results and final approval of the version to be published; JR Velázquez-Martínez, conception, design, critical review for important intellectual content, interpretation of results and final approval of the version to be published; M Torres-De la Cruz, design, critical review for important intellectual content, interpretation of results and final approval of the version to be published; A De la Cruz-Pérez, design, critical review for important intellectual content, and final approval of the version to be published; S Capello-García, design, critical review for important intellectual content, and final approval of the version to be published; MA Hernández-Gallegos, critical review for important intellectual content, and final approval of the version to be published.