Response of the subterranean termite Reticulitermes grassei Clément (Isoptera: Rhinotermitidae) to pH of substrate

Journal Pre-proof ´ Response of the subterranean termite Reticulitermes grassei Clement (Isoptera: Rhinotermitidae) to pH of substrate ´ A.M. Cardenas, P. Gallardo, J.R. Carbonero-Pacheco, M. Trillo

PII:

S0031-4056(19)30269-0

DOI:

https://doi.org/10.1016/j.pedobi.2019.150608

Reference:

PEDOBI 150608

To appear in:

Pedobiologia - Journal of Soil Ecology

Received Date:

29 July 2019

Revised Date:

25 November 2019

Accepted Date:

25 November 2019

´ Please cite this article as: Cardenas AM, Gallardo P, Carbonero-Pacheco JR, Trillo M, ´ Response of the subterranean termite Reticulitermes grassei Clement (Isoptera: Rhinotermitidae) to pH of substrate, Pedobiologia - Journal of Soil Ecology (2019), doi: https://doi.org/10.1016/j.pedobi.2019.150608

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Response of the subterranean termite Reticulitermes grassei Clément (Isoptera: Rhinotermitidae) to pH of substrate A.M. Cárdenas*, P. Gallardo*, J.R. Carbonero-Pacheco*and M. Trillo*

*Department of Zoology, Edif. Darwin, Campus Rabanales, University of Córdoba, Córdoba E-14071,

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Spain Correspondence: A.M. Cárdenas; e-mail: [email protected];

Highlights

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Soil pH as control methods to R. grassei (ISOPTERA) R. grassei is tolerant to basic substrate Alkalization of substrate would not be a feasible procedure to control R. grassei populations in forest of southern Iberian Peninsula.

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Abstract

Reticulitermes grassei Clément is an Iberian subterranean termite whose feeding activity can damage both urban infrastructures and oak forests, becoming a main pest. In consequence, the development of

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new control methods compatible with the environmental conservation is a priority. Recently, techniques for termite control supported on the fact that the soil pH can be a chief factor in restricting termite populations have been explored. On this basis, we consider the possibility of taking advantage of certain

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current harmless silvicultural techniques, which also vary the soil pH, to control populations of R. grassei in natural environments. Before addressing this question, it was necessary to know the termite response to pH of substrate.

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For this instance, this work aimed to establish the range of pH in different zones of R. grassei nests and to determine the tolerance of this species to pH ranges outside of those observed in field. In addition, the effect of termite activity on soil pH is assessed. This research has been carried out from October 2018 to March 2019, in several natural areas from Córdoba (Spain). The results indicate that R. grassei preferably colonizes neutral or somewhat acid substrate, ranging from 5.4 to 7.5; the pH values in the inner nest increases towards the periphery. In addition, this species shows rather tolerance to alkaline substrate (pH≤8.5). Nevertheless, it seems to be more sensitive to acid pH, being observed the lowest tolerance limit at pH≈ 4.5; if pH drops to 4.0, mortality is near 100%. According to our results, the

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alkalization of substrate would not be a feasible procedure to control R. grassei populations in forest of southern Iberian Peninsula.

Keywords: Isoptera, pH, Reticulitermes grassei, Rhinotermitidae, soil fauna, subterranean termites Introduction Reticulitermes grassei Clément is a subterranean termite extended in northern, western and southern Iberian Peninsula and southern-west France (Clément et al., 2001). This termite lives and reproduces in

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the soil and feeds mainly on wood (Lenz et al., 2003), causing damage to urban infrastructure, thus becoming a major pest. The number of affected buildings in certain areas of Andalusia (southern Spain) reaches 33% (Gaju et al., 2002). Like other subterranean termites, R. grassei also colonizes natural environments, where it plays an important role contributing to natural recycling by eating wood and soil (Pinzon et al., 2006). Regarding the environmental factors influencing presence and seasonal activity of

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this termite, overall, the species could be considered eurytopic, showing higher forest activity at the early autumn, ending September, “after the first rains” (Cárdenas et al., 2012, 2018). Although, this

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species is very common in most Iberian forests, its presence had not been considered a pest. Recently, in cork-oak forests (Quercus suber L) from Andalusia, were observed lesions caused by the feeding activity performing sinuous galleries and making adhesions. These lesions hinder the normal cork’s

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extraction (Gallardo et al., 2010), devaluating the product and threatening the sustainable exploitation of these forests (Cárdenas et al., 2012).

Among the most standardized procedures to control this pest in urban environments are the chemical

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barrier systems which work by keeping the termites out with the use of repellents (Bifenthrin) or relying on the termites taking the bait back to the colony to destroy (Fipronil) (Vargo & Parman, 2012; Rust, 2014). These types of substances are highly toxic to other non-target insects making it impractical to be

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used in natural ecosystems (Gant et al., 1998; Van der Sluijs et al., 2015). The use of this type of insecticide is being increasingly restricted by the European legislation (EU Commission Regulation n°

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1127/2014), resulting the development of new control methods compatible with the environment a priority (Mahapatro & Madhumita, 2014). Recently, new trials for termite control have been performed (Neupane et al., 2015; Li et al., 2017). There are supported on the observation that soil pH can be a chief factor in restricting presence or the population size of certain termite species such as Odontotermes formosanus and Reticulitermes flaviceps. In fact, targeted application of salt dissolutions in soils has been already tested in Zhejiang province of China (Chen, 2002; Chen et al., 2011). Although pH is a crucial variable involved in the soil biological processes, its effect on soil dwelling organisms is little known and research is mostly focused on annelids Lumbricidae (Graefe & Beylich,

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2003). Concerning other edaphic fauna, it is known that nematodes are more common in soils with high pH, while the abundance of arthropods is negatively correlated with this parameter (Wu et al., 2011; Zhao et al., 2017), so that the response to edaphic variables, including pH, may be different depending on the arthropod Orders (Zhao et al., 2017). Focusing on Isoptera, different effects on soil pH has been observed depending on the species and the soil type (Neupane et al., 2015). In most of the cases, this parameter seems to be increased by the termite activity (Nutting et al., 1987; Donovan et al., 2001; Robert et al., 2007; Neupane et al., 2015). Regarding Reticulitermes species, it has been found that R. flavipes rises or decreases soil pH depending

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on the presence or absence of wood (Neupane et al., 2015), and that while it shows preference by acidic environment, its activity results in basification of substrate (Li et al., 2017).

Bearing in mind prior information, we consider the possibility of taking advantage of certain silvicultural techniques, usually addressed to others phyto-sanitary problems (i.e.root mycosis; LealMurillo et al., 2012), and that also act varying soil pH, to control the damage of R. grassei in natural

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environments. Concretely, in “dehesas” of the south of the Iberian Peninsula, limestone amendments with different calcium salts (calcium oxide, calcium carbonate and calcium sulphate) are applied to

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control the root rot of Quercus sp. caused by the oomycete Phytophthora cinnamomi and Pythium sp. (Serrano et al., 2013). In addition, calcium amendments are used to palliate the effect of the acid rain (Monfort-Salvador et al., 2015) and to neutralize the acidity and to reduce the exchangeable aluminum

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in acid agricultural soils (Elisa et al., 2016).

Before performing tests aimed to investigate the effect of soil amendments on the R. grassei populations, it is necessary to investigate the response of this subterranean termite to edaphic pH

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variations.

For this reason, this research addressed three main objectives: I.

To establish, in field, the range of pH in the different zones (inner, foraging area and external

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zone) of the R. grassei nests.

To determine the tolerance of the insects to pH ranges outside of the observed in its natural

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environment. III.

To assess the effect of termite activity on the soil pH under controlled conditions.

Methods

pH determination in Reticulitermes grassei nests To determine the range of soil pH suitable for R. grassei, 16 nest were surveyed (Table 1). Six of them were in the surrounding of the Rabanales Campus, University of Córdoba (Spain). This area shows a high anthropization level and lodges several buildings, sport facilities and infrastructure diverse.

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Five nests are situated in the "Román Pérez" farm, a natural space next to the university Campus, in the foothills of Sierra Morena, intended to carry out environmental studies. The remaining five nests are situated in Central Sierra Morena, in the mountain farm “Los Baldíos” where considerable damages by R. grassei have been described in cork oaks (Gallardo et al. 2010). In “Román Pérez” and in “Los Baldíos” the main vegetation consists of a mixed oaks forest of great ecological value, Quercus ilex, Q. suber and Q. faginea are the tree species dominants, while Pistacia lentiscus, Asparagus albus, Arbutus unedo and several species of Erica and Cistus are the species integrating the scrub (Torres & Ruiz,

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2009). Three sampling zones were established in each nest examined (Fig.1) according to the presence of different castes of termites (see Bordereau et al., 2002 for castes definition): The inner nest (IN), where specimens of all the castes can be found; the foraging area (FA), where soldiers and workers are only present, and the external zone (EZ), where only is detected occasional presence of workers, and

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very seldom soldiers.

In autumn 2018, coinciding with the maximum surface activity of R. grassei observed in field (Cárdenas

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et al., 2012), two measures were taken and averaged at each one of the three zones of the 16 nests. After moistening the soil with distilled water, the pH was measured with a portable soil pH-meter (PCE-

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PH20S).

Response of Reticulitermes grassei to pH of substrate

The termites were collected in field, between September and October 2018. Four stations baited with

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corrugated cardboard (Gallardo et al., 2010) were placed next to the nests located in the university Campus. The baits with termites were weekly removed; the baits were replaced, and the termites were taken to the laboratory to be reared in a steel container (of 1x1x 1m3) provided with trunks of Pinus

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pinea L. for insect’s feeding and accommodation. This wood species is considered the most suitable for laboratory studies with subterranean termites (Perkins, 2012; EN 118, 2013).Termites were kept in a

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rearing container, under controlled conditions of permanent darkness, 28±1 ºC temperature, and 80 ± 5% relative humidity, until to be used for trials. To determine the range of tolerance of R. grassei, experiments with pH ranging from 4.0 to 8.5 were performed (excepting values comprised into the range of the species in field). We studied the response to pH values 4.0, 4.5, 8.0 and 8.5. In addition, one experiment with substrate of pH=7.3 was also performed as control. Benzoic acid for pH ≤ 4.5 and calcium carbonate for pH ≥ 8.0 were used to obtain different pH values of substrate. Benzoic acid was selected because it is a degradation intermediate product of lignin’s digestion (Geib et al., 2008) being present in the gut of these insects, calcium carbonate is part of the

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most of natural substrates where R. grassei live in field. Both products are harmless to the termites. Different adjustment tests were performed, dissolving each product in distilled water prior to be mixed with the vermiculite, until obtaining the pH required. Final concentrations of dissolutions added to the vermiculite, for each pH value, are: pH 4.0 (10 ml C6H5-COOH 0.012 M); pH 4.5 (10 ml C6H5-COOH 0.008 M); pH 7.3 (10 ml distilled water); pH 8.0 (10 ml CaCO3 1 M); pH 8.5 (10 ml CaCO3 1.5 M). For the trials, small containers (11.5 cm Ø x 6 cm height) were used, containing 14 g of vermiculite as substrate and five blocks of approximately 2x2x1 cm3 of P. pinea. The insects were extracted, manually and carefully, from the rearing container. Later, it was checked that the workers coming from the same

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colony and that were at least of the third instar (Chouvenc et al., 2011). A total of 150 workers were placed into each trial container and were kept at the same initial conditions. Three exposure times to the different pH values were applied: 15, 30 and 45 days. For the follow-up, once the respective checking time were elapsed, the survivor termites were counted.

There were five trial containers (repetitions) per pH experienced and exposure time (total 75

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repetitions). In addition, there were five repetitions per pH without termites which were used to follow

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pH variations at 15, 30 and 45 days, in the same container (25 repetitions; Figure 2).

Effect of Reticulitermes grassei in the pH of substrate

To know the effect of the termite activity in the pH of substrate, data relative to pH obtained after 15, 30

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and 45 days in the experiments conducted at different pH values referred in previous section were

Data analysis

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analysed.

Analysis of variance (one-way ANOVA) were used to test the differences in the averaged pH values of the three zones of the termite nests, to determine the effect of termite activity on the pH of substrate and

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to discern differences in survival vs. time of exposure (15, 30 and 45 days) and vs .pH values (4.0, 4.5, 7.3, 8.0 and 8.5). Assumptions of normality and homoscedasticity were checked with Shapiro-Wilk

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test and Lèvene test, respectively (Zar, 1999). If data did not satisfy these criteria, the nonparametric Kruskal-Wallis test was applied instead. The post-hoc Tukey-Kramer test or the diagrammatic box plots were performed for the ANOVA or the Kruskal–Wallis test, respectively. To assess the tolerance to pH the survival rate was calculated. Survival rate is the percentage of surviving termites at the end of each trial. Obviously, if mortality of a concrete trial was ≈ 100%, the tolerance results zero and the respective pH value was not included in the analysis of results. To explore the effect of R. grassei in the pH of substrate, comparisons of pH in trials with and without termites were performed using paired Student t-tests. If normality assumption was not satisfied, the equivalent non-parametric Mann-Whitney U test was performed. For each pH experienced, averaged

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values with termites and their respective control without insects (at 15, 30 and 45 days) were compared. In addition, pH variations on time in different control trials were checked by applying the one-way ANOVA. Probability for tests signification was α=0.05 and calculations were performed using SP statistical software (SPSS Inc., 2011).

Results pH determination in Reticulitermes grassei nests

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The pH values recorded in the different zones of each sampling nest were averaged, and the results are shown in Table 2.The pH values in the inner nest are lower than in the foraging area and external zone, with increasing trend from the interior to the periphery. These differences were statistically significant (Kruskal-Wallis test: χ²=26.89; P<0.05). The pH value of the substrate for R. grassei in field ranged

Response of Reticulitermes grassei to pH of substrate

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from 5.4 to 7.6, ( =6.48 ±0.68 inner nest; =7.06±0.40; foraging area; =7.23±0.34 external zone).

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The percentage of survivors in the experiments carried out at different pH values and exposure times are displayed in Table 3. It is noticeable the high overall survival, over 80%, obtained in all of the pH experienced except for pH=4.0, in which the average survival only reaches 3.56%.

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This remark is confirmed after making the pertinent statistical analysis (Kruskal-Wallis test: χ²=48.16; P<0.05). By repeating the comparative analysis but discarding the experiment at pH=4.0 which obviously generate the major discrepancy, significant differences are again obtained (Kruskal-Wallis

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test: χ²=19.67; P<0.05).

The respective box- plots (Fig. S1) indicate that the best results in terms of survival correspond to the

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trial performed a pH=8.0.

By breaking down the joint analysis according to the time of exposure to each pH value on the survival

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rate of the termites, the statistics find significant differences in all cases, whether the results corresponding to the trial performed at pH=4.0 are included or not (Table 4).

Analyzing the effect of exposure time to each pH value on the survival rate of termites, at pH similar to the external area of the nest (pH=7.3), the statistic did not find significant differences after 15, 30 and 45 days of exposure (ANOVA test: F=2.33; P> 0.05). When the follow-up is performed at pH values out those the field range observed for R. grassei, the results are:

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1. Basic substrate: proceeding in a similar way but with the data from the experiment conducted at pH= 8.0, significant differences (ANOVA test: F=4.46; P< 0.05) are remarked. The post-hoc Tukey test allows discerning that differences are among the survival rate recorded after 15 days and those corresponding to 30 and 45 days (Table S1). Similarly, at pH=8.5, the Kruskall-Wallis test showed in terms of survival between the results obtained after 15 days and the survival recorded later in the follow-up, but not between the number of survivors after 30 and 45 days exposure (Kruskal-Wallis test: χ²= 8.64; P< 0.05; box-plots in Fig. S2). 2. Acid substrate: At pH=4.5 there were not significant differences in survival over time (ANOVA test:

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F=2.23; P>0.05), but in the trial at pH= 4.0, after 15 days the survival rate was very low (10.6%) and after 30 days of exposure, there were no survivors. Effect of Reticulitermes grassei in the pH of substrate

Although minor fluctuations were observed, the statistic test did not find significant differences

(ANOVA test: F=0.68; P> 0.05), thus indicating that the pH of the substrate remained stable during the

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45 days of follow-up.

Nevertheless, in a similar trial but with termites there was a downward trend as time went by, which was

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ratified statistically (ANOVA test: F=33.44; P< 0.05). The post-hoc Tukey test allows discerning that differences are among the pH recorded after 15 days and those corresponding to 30 and 45 days (Table S2).

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First, variations over time in the control at pH=7.3 were checked (Table 5).

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Regarding paired comparisons of pH with termites and its respective controls at different exposure times (Table 6), in the trial at pH=7.3, after 15 days no differences are produced, but in trials of 30 and 45 day

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the pH values were significantly lower in trial with insects.

The analysis of the results obtained outside the pH range in field for the species reveals that:

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1. Basic substrate: At basic pH=8 and pH=8.5 (Table 5) there are no significant differences over time in the control (ANOVA test: F=2.602; P> 0.05; Kruskal-Wallis test: χ²=9.5; P> 0.05, respectively), but in the respective trials containing insects the pH varied significantly (Kruskal-Wallis test: χ²= 8.79; P< 0.05; and ANOVA test: F=17.38; P< 0.05, respectively). These differences at pH=8 were graphically discriminated by the box-plots (Fig. S3). Initially the pH rose next to 8.5 but at the end of the experiment it was stabilized at pH similar to the starting value. In trials at pH=8.50 with termites, once 15 days elapsed, significant differences were obtained for any exposure time (30 and 45 days; Table S3; post- hoc Tukey tests).

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Statistical paired comparisons in trial with termites vs. control for pH=8.0 found significant differences after 15 and 45 days the follow-up (Table 6); and for pH=8.5 significant differences were got at any exposure time. 2. Acid substrate: in the controls at pH=4.5 and pH=4.0 (Table 5) there were significant differences over time (Kruskal-Wallis test: χ²=12.5; P< 0.05; ANOVA test: F=22.78; P< 0.05, respectively). In trials with termites the pH of substrate also varied significantly (ANOVA test: F=17.84; P< 0.05 for pH=4.5). Differences in control at pH=4.5 were discerned by the post-hoc Dunnet T3 test (Table S4) showing that during 15 days the pH remained quite similar to the starting value, but after 30 and 45 days this

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parameter rises reaching 6.0. The trial with pH=4.0 shows the same trend to increase, reaching the maximum values once elapsed 45 days (Table S5; post-hoc Tukey tests). In presence of insects, increasing pH is observed for 15 days exposure, reaching 7.5 at the end of the experiment (Table S6; post- hoc Tukey tests). In trials performed at pH=4.0 and higher times of exposure, mortality was near 100%. So, the course of pH on these trials was not considered. Statistical paired comparisons in trial

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with termites vs. control for pH=4.5 found significant differences after 15, 30 and 45 days exposure (Table 6). For initial pH of 4.0, comparison only was calculated after 15 days exposure. The statistic

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“T” test found significant differences between the trials with and without termites (Table 6).

Discussion

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Nowadays research on restricting presence of damaging termites is a priority. Nevertheless, the harmful effect of the continued use of chemical termiticides is a serious concern so that many of the most effective chemical agents are now banned under more compromised environmental regulations. In

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consequence, developing management strategies for termite control avoiding environmental deterioration are a crucial subject (Mahapatro & Madhumita 2013, 2014). Indeed, it arises the need to initiate new lines of research that allow finding alternatives for the appropriate management of those

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termite species potentially pest without disturbing the environment but ensuring the soil biota performance.

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Under this context, our research addresses relationships between the pH of the substrate and the activity of the termite R. grassei, as fundament to exploring of new control procedures. The main results obtained are that this species colonizes neutral or slightly acid soils, shows well tolerance to alkaline substrates, but pH values below 4.5 are not suitable, becoming lethal at pH= 4.0. The response in terms of pH tolerance of other termites is rather variable. Studies with diverse species and geographical areas show that some of them colonize quite acid soils; for example, for Subulitermes microsoma or Anhangatermes macarthuri, inhabitants Central Amazonia, the pH was around 4.3-4.4 (Ackerman et al., 2007). In contrast, the subterranean termites Heteritermes aureus and Gnathamitermes perplexus seem to be favored in more basic substrates and they increase the soil pH

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from 6.8 to 7.9 and 7.6, respectively (Nutting et al., 1987). Trend to colonize neutral or slightly acidic environments can be also found: Afolabi et al. (2014) indicate that species from Nigerian savanna, as Macrotermes bellicosus and Trinervitermes geminates live in soil with pH around 6.5. Similar trend has been referred for other species (Constantino, 2002; Davies et al., 2003; Wink et al., 2005; Lavelle et al., 2006; Acda, 2013), and for R. grassei in this work. In terms of range of pH tolerance, measured also in nature, some authors (Li et al., 2017) point to high tolerance in the case of O. formosanus and R. flaviceps, species found in substrates markedly acidic (pH≈ 3.5) and in those of significant alkalinity (pH ≈ 8.7). These authors review the pH of the nest and

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surroundings areas of 117 cases studied, concluding that most of them nest in acidic or feebly alkaline substrates while the surrounding soils were acidic. This implies that most termites choose acid soils but into the nests the pH is usually somewhat higher. This outcome is contrasted to our observations for R. grassei since the lower pH values correspond to the inner nest. Differences in pH recorded at different areas of the nest for R. grassei, agree with those are stated Batalha et al. (1995) and Ackerman et al.

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(2007) who found significant differences among the areas of the termite mound. Accordingly, with Hopkins et al. (1998), Ji & Brune (2005) and Brune (2014) it could be argued that, joint to other factors,

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in the pH of the nest intervene the digestive biochemistry of these insects, feeding mainly of wood and vegetal detritus. The feces produced contain high content of lignin, acetate and other short chain compounds which are released in soil, going down pH, particularly in those areas of the nest with

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highest concentration of individuals such as the inner nest, where wastes are accumulated. Other reasons could also explain the observed differences: Davies et al. (2003) suggested that the high presence of termites in areas with slightly acidic pH values may be a compensation mechanism, since

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digestion increases the intestinal pH. Other authors also point that it may be a specific character (Dhembare, 2013), or to an adaptive response to a latitudinal gradient, being the species from temperate zone more sensitive to acid soils than those from tropical forest (Briones, 2014). In these areas,

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lixiviation due to precipitation affects soil characteristics, including pH (Quinto-Mosquera & MorenoHurtado, 2016). In addition, the effect of termites on water and soil nutrients drainage through leaching

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(Rückcamp et al., 2009) induce to the soil acidification to which termites must be adapted. Minor adaptability to low pH in temperate zone is consistent with our results, since R. grassei was no tolerant to pH ≤4.0.

Dealing with a taxonomically closer species to R.grassei, as it is the case of R. flavipes, Neupane et al. (2015) argued that the occurrence of wood alkalinizes the soil while the termite presence acidifies it. The same authors suggest that acidification may be the result of the decomposition of organic matter and of the own secretions of the insects in the foraging areas. These processes may explain that in our trials with alkaline substrate the pH was lower in the presence of the insects than in the respective controls.

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Analyzing the inverse effect, that is, how soil pH can restrict the presence of R. grassei? Until now no research had addressed this issue. From the high overall survival (over 80%) obtained in most our trials even in those performed out of the range for the species in field, it could be catalogued as eurytopic to this parameter. The lowest tolerance limit was observed at pH≈ 4.0, because in the corresponding trial, the average survival only reaches 3.56%; after 15 days of exposure little more than 10% of termites survive, and after 30 days mortality was 100%. Comparing results from neutral and alkaline trials, it was observed that survival becomes favored with slightly alkaline substrate, but this tendency is unappreciable to more basic values (pH =8.5).

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In addition, it should be considered that the diet of subterranean termites is nutritionally poor, but these organisms need the same basic nutrients as the rest to develop the processes of cellular metabolism (La Fage & Nutting, 1978). Nevertheless, there are few studies addressing the question of how termites get the microelements that they need (Ca, Fe, Mg or Mn), (Janzow & Judd, 2015). For this reason, we hypothesized that the benzoic acid or the calcium carbonate added to the substrate could be ingested by

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the termites along with moisture, enriching their diet. Related with this issue, it could be discussed if the substances that we added to the substrate in order to obtain the pH selected for trials could nutritionally

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affect the termites, and therefore, the survival obtained. Of course, that both benzoic acid and calcium carbonate are harmless for termites and that for this reason we select them to perform the tests, but what other possible effect could they produce? In this sense, no data are available regarding R. grassei. The

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effect of calcium on feeding selection has been studied in a close species, R. flavipes, with the basis that calcium is an important micronutrient for metabolic pathways and cell growth (Frausto da Silva & Williams, 2001). The results showed that R. flavipes does not prefer diet calcium enriched but this

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element may affect the foraging strategy since the different castes requires specific nutrients for growth and reproduction. This matter did not affect our study since we did not work with the nest as a whole, and only workers were used. Concerning the addition of benzoic acid, the aromatic compounds

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derivatives of the benzoic acid are usually considered attractants for Reticulitermes species (Peterson, 1998). Chromatographic studies show that among the main lignin-derived compounds present in termite

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nests are aromatic moieties as 3, 5-dihydroxybenzoic acid which is also produced during cupper oxidation of plant tissues (Vane et al., 2013). This compound should not have any effect in our study because in wood supplied as food there are multiple precursors of this substance such as tannins and aromatic compounds (Vane et al., 2001). Finally, we addressed the question whether soil basification treatments could provide an additional benefit by controlling the populations of R. grassei in Mediterranean oak forests. Our hypothesis was supported in the observations of Li et al. (2017), who indicated that areas with soil pH from 8.12 to 8.94 were free of termites and that increasing pH around 9.5 may induce the inactivation of this insects. In

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practice, theses authors tested, in China, whether irrigation with seawater could be used for termite control. By amending the proposal to our case, soil improvement with calcite addition is a very common practice because calcium increases the tolerance of Q. ilex to the Phytophthora root disease affecting oak rangeland ecosystems (Serrano et al., 2013). Amendments with calcite raise the pH of soil normally one unit (Espinosa & Molina, 1999). According to the “Typology of soils in the Andalusia community” (https://www.juntadeandalucia.es/medioambiente), the forests where damages by R. grassei were recorded correspond to soils with neutral or slightly acid pH and low in calcium content. By adding

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calcite, the soil pH will raise until 8.0-8.5, but from our experiments R. grassei may be considered as good tolerant to basic pH (≤8.5).

Conclusions: in view of our results, the natural range of soil pH in field for R. grassei oscillates between 5.4 and 7.4 and varies in the different zones of the nest, showing a trend to increase from the center of to the periphery; the insect’s foraging activity raises the substrate pH up to alkaline values; and

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regarding the survival rate this species show enough tolerance to basic substrates.

In addition, we conclude that soil alkalization by calcite amendments seems not be a feasible procedure

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to control R. grassei in forests of the southern Iberian Peninsula. More research is necessary to explore new control methods based on the restrictive effect of soil pH on the termite populations.

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Acknowledgments

We thank to PhD. García-Núñez (Organic Chemistry Dept., Córdoba Univ. Spain) her assistance in the

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pH adjustments and to Prof. Sutcliffe the language revision.

Conflicts of interest: The authors declare no conflict of interest.

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Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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Zhao, K., Jing, X., Sanders, N.J., Chen, L., Shi, Y., Flynn, D.F.B., Wang, Y., Chu, H., Liang, W. & He, J.S. (2017) On the controls of abundance for soil-dwelling organisms on the Tibetan Plateau. Ecosphere

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8(7), 1-14.

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17

4.0

5

4.5 CONTROL TRIALS

8.0

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8.5

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4.0

TRIALS WITH TERMITES

X

X

X

5

5

5

5

X

X

X

5

5

5

5

5

X

X

X

5

5

5

5

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7.3

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5

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Figure 1 Schematic representation and dimensions of the areas surveyed in each nest of R. grassei. IN: Inner nest; FA: Foraging area; EZ: External zone. (Values correspond to arithmetic mean ± standard deviation). pH REPLICATES CHECKING TIME (days) TRIALS VALUES NUMBER 15 30 45

5

X

X

X

5

5

5

5

5

X

X

X

5

5

5

5

5

X

5

5

5

X

5

5 X

5

5

5 4.5

5

X

5

5

5

X

5

5 X

5

5

5 7.3

5

X

5

5

5

X

5

5 X

5

5

5 8.0

5

X

5

5

5

X

5

5 X

5

5

5 8.5

5

X

5

5

5

X

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18

Figure 2 pH values experienced in the control trials (without termites) and in the trials with termites;

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number of repetitions performed (replicates number); and days elapsed for pH follow-up (checking

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time).

19

Table 1 UTM Coordinates corresponding to the surveyed nests of Reticulitermes grassei.

30S 0348394 30S 0348735 30S 0348732 30S 0349097 30S 0349266 30S 0379133 30S 0335275 30S 0335422

Nest reference

4197822 4197822 4197825 4197780 4197863 4710295 4200874 4201107

9 10 11 12 13 14 15 16

UTM Coordinates 30S 0335411 30S 0335417 30S 0335433 30S 0349301 30S 0349304 30S 0349301 30S 0349300 30S 0349298

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1 2 3 4 5 6 7 8

UTM Coordinates

4201107 4201111 4201013 4201047 4201022 4201099 4200933 4200978

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Nest reference

20

Table 2 Averaged pH values obtained in the three zones of the nest (IN: inner nest; FA: foraging area; EZ: external zone; : Mean; SD: Standard Deviation). Nest Reference

IN SD

FA SD

EZ SD

IN SD

Nest Reference

FA SD

EZ SD

5.73±0.06 6.90±0.10 7.43±0.06

9

7.23±0.06 7.30±0.07

7.07±0.09

2

5.87±0.15 7.03±0.06 7.20±0.26

10

7.41±0.02 6.97±0.06

7.43±0.22

3

5.93±0.12 6.77±0.06 7.33±0.21

11

7.30±0.35 7.07±0.10

7.04±0.04

4

5.93±0.06 7.20±0.10 7.23±0.06

12

6.89±0.04 6.91±0.13

7.17±0.06

5

5.43±0.06 7.27±0.21 7.43±0.06

13

6.53±0.21 6.66±0.05

6.51±0.03

6

6.73±0.06

7.37±0.12

14

6.87±0.18 7.24±0.13

6.74±0.17

7

7.54±0.13 7.77±0.09 7.67±0.01

15

6.83±0.12 7.64±0.06

7.87±0.15

8

7.09±0.04 5.85±0.16 6.97±0.04

16

5.96±0.04 7.19±0.21

6.82±0.18

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7.20±0

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1

21

Table 3 A) Overall Survival Rate: mean of the percentage of survivors recorded for the whole of the experiment for each pH value tested. B) Survival Rate: mean of the percentage of survivors recorded at different pH value of substrate and exposure time, in days ( : arithmetic mean; SD: Standard Deviation). Freedom degree = 4. A) Overall Survival Rate

SD

93.02

95.11

86.80

4.01

3.46

9.35

B) Survival Rate

pH = 7.3 pH = 8.0 pH = 8.5 :

93.87

98.93

SD

3.31

1.53

89.60

93.87

2.56

1.97

SD

95.60

45

3.85

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na

SD

3.56

5.99

13.77

pH = 4.5

pH = 4.0

91.47

10.67

5.11

23.85

93.73

84.80

0

3.55

6.40

0

92.53

88.93

82.80

0

2.69

3.08

2.64

0

re

30

77.73

86.36

10.75

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15

pH = 4.0

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Time

pH = 4.5

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pH = 7.3 pH = 8.0 pH = 8.5

22

Table 4 Statistical comparison of the survival rates (mean of the percentage of survivors) recorded at different values of pH for each of exposure time (in days). A) Including all the trials performed. B) Excluding the trial performed at pH=4.0 (χ²: values of non-parametric Kruskal-Wallis test; F: values of one-way ANOVA test; P: probability). A)

B)

Statistic

P

Time

15

χ²=20.28

0

15

χ²=13.58 0.004

30

χ²=18.36 0.001

30

χ²=10.56 0.013

45

χ²= 0.27

45

F=15.74

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0

Statistic

P

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Time

0

23

Table 5 Averaged pH values of substrate obtained at different exposure times (in days) in trials without termites (Control) and with termites (Termites) (

Arithmetic Mean; SD: Standard Deviation).

Freedom degree = 4. Time Trial

7.3 Termites Control

15

30

45

7.30

7.53

7.70

7.56

0.11

0.34

0.23

7.30

7.61

6.65

6.77

0.10

0.10

0.24

0.23

8.00

8.38

8.24

8.33

0.06

0.08

0.12

8.46

8.30

8.14

0.05

0.15

0.04

8.60

8.40

8.40

0.03

0.10

0.05

8.50

8.43

8.17

8.30

0.05

0.09

0.03

4.62

5.20

5.97

0.09

0.19

0.57

4.50

5.94

7.21

7.61

0.37

0.56

0.42

4.09

4.13

4.50

0.08

0.10

0.12

5.07

--

--

0.43

--

--

SD SD SD

8.0

8.00 Termites

SD SD

8.5 Termites

-p

8.50 Control

SD

4.50

4.5 Termites

SD

lP

Control

SD

4.00

4.0

ur

Termites

Jo

SD

na

Control

SD

ro of

Control

1

re

pH

4.00

24

Table 6 Statistical paired comparisons of averaged pH of substrate with termites and its respective controls at different exposure times (in days) and pH values (T: Student T test; Z: Mann-Whitney U test, P: probability).

7.30

15

8.00

T/Z

P

Time

T/Z

P

Time

T/Z

P

-1.30 0.230

30

5.56

0.001

45

5.43

0.001

15

-2.33 0.048

30

-0.75 0.473

45

3.03

0.035

8.50

15

2.61

0.008

30

3.57

0.007

45

3.37

0.010

4.50

15

-7.68 0.001

30

-7.49 0.001

45

-5.14

0.001

4.00

15

-4.98 0.006

--

--

--

--

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Time

--

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re

-p

pH

--