Introduction

Gossypium hirsutum L. (Cotton), a vital fiber crop cultivated in regions spanning latitudes 37° North and 32° South. It serves as a cornerstone of global commerce, generating substantial income for producing nations and bolstering local economies (Basal et al., 2019). Cotton cultivation also plays a significant role in the Turkish economy by contributing substantially to the nation’s export revenues. It is mainly concentrated in regions such as the Aegean, Southeastern Anatolia and Mediterranean (Cevheri & Şahin, 2020).

However, ecological challenges posed by global warming, as well as important pest species prevalent in production areas, are significant factors limiting cotton production. Spider mites are one of the most important group of cotton pests in the world (Guo et al., 2013; Jeppson et al., 1975; Sulek & Cakmak, 2022). These mites are polyphagous pests that can be found on various parts of the cotton plant, particularly on the undersides of young and vigorous leaves (Jeppson et al., 1975; Migeon & Dorkeld, 2024). They often form high density populations at the axil between the leaf petiole and leaf blade and can spread to other parts of the plant through the webbing they produce while feeding. Their feeding activity results in characteristic scattered yellow spots on the upper leaf surface. Over time, the yellow spots turn red due to damage to the chlorophyll, which gives the leaf its green color. This reddening intensifies, ultimately causing premature leaf desiccation and abscission. High densities of spider mites can also lead to the shedding of bolls, flowers, and small boll sizes (Jeppson et al., 1975; Simons, 1964; Sulek & Cakmak, 2022; Sulek et al., 2023).

Tetranychus cinnabarinus Boisduval [synonym of T. urticae Koch (Auger et al., 2013)], T. atlanticus McGregor (currently known as T. turkestani (Ugarov & Nikolskii); Migeon & Dorkeld, 2024), and T. desertorum Banks have been identified as the dominant mite species in cotton fields in Türkiye (Düzgüneş, 1962; Dinçer, 1975). Over the years, these spider mites have posed a significant threat to cotton-growing areas within the Aydin province, typically between late April or early May until harvesting in September and October. Cotton growers have reported an increase in spider mite problems and have encountered challenges in effectively managing these pests (Sulek & Cakmak, 2022; Sulek et al., 2023). The difficulty in control may arise from the presence of different Tetranychus species or the development of resistance to acaricides by the spider mite population. Preliminary studies have found T. urticae in a few cotton leaf samples (Sulek & Cakmak, 2022). However, other spider mite species, such as T. atlanticus or T. desertorum, may also be present in cotton fields, making control more challenging (Düzgüneş, 1962; Dinçer, 1975). The last comprehensive study on Tetranychus species diversity in the region was conducted by Dinçer (1975). Therefore, it is necessary to re-evaluate the spider mite species found in cotton fields of Aydin province, specifically in cotton growing areas of Soke, Germencik, Kocarli, and Nazilli through both morphological and molecular species identification. Such studies would provide important insights into the species composition and distribution of spider mites in Aydin province. Additionally, they would help reveal the presence of associated endosymbiotic bacteria and guide future research in this area.

Pesticide degradation by certain microorganisms within the arthropods’ microbiome is another mechanism of resistance development. The role of such arthropod microbiota in many physiological and metabolic functions has emerged as an important area of study (Zhu et al., 2019). Endosymbiotic bacteria, such as Wolbachia and Cardinium, are known to be present in arthropod hosts, including mites (Hoy & Jeyaprakash, 2005; Enigl & Schausberger, 2007; Pekas et al., 2017; Zélé et al., 2020). These endosymbiotic bacteria have been reported to promote parthenogenesis, induce feminization of populations, cause male mortality, and lead to cytoplasmic incompatibility. Wolbachia has also been found to protect against viral infections in its hosts. Although the effects of Wolbachia and Cardinium on their hosts are not yet fully understood (Ros et al., 2012), it has been suggested that some endosymbiotic bacteria can provide defense mechanisms against natural enemies and degrade pesticides (Güz et al., 2015). Understanding the mechanisms of microbiota-mediated resistance could lead to novel control strategies that manipulate or target the spider mite microbiome.

The main objective of this study is to determine the species diversity of Tetranychus mites in cotton-growing areas of Aydin province through morphological and molecular species identification. Additionally, it aims to determine the endosymbiotic bacteria associated with these mites. These efforts will provide important information about the species of spider mites and their distribution in Aydin province, as well as contribute to understanding the potential impacts of endosymbiotic bacteria on the development of resistance to acaricides in this region.

Material and methods

Sampling

Samples were collected from the districts of Germencik, Kocarli, Nazilli and Soke which are the largest cotton producers in the Aydin province in June, July, and August of 2020–2021. Leaves infested with spider mites (~ 20 leaves/field) were collected with the aid of hand lens (× 30) from cotton fields in each district separately. A total of 220 fields (2–50 hectares) were sampled (Fig. 1), and the colors of spider mite samples were also recorded. During sampling, cotton leaves were initially placed in paper bags and then transferred to polyethylene bags. Subsequently, the samples were stored in an ice box and transported to the Acarological Laboratory of Aydin Adnan Menderes University. The samples were kept in a refrigerator (4 °C) until examination. For molecular identification of Tetranychus species, 100 adult females from each field were individually placed into microcentrifuge tubes using a stereo microscope (Leica EZ4) and a vacuum pump (KNF N022 AN18). To prevent contamination between sampled areas, hands and arms were cleaned with 96% ethyl alcohol before collecting samples from any other field.

Fig. 1
figure 1

Sampling sites in 2020 and 2021

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Morphological identification of Tetranychus species

Adult females and males were directly mounted ventrally and dorso-ventrally, respectively, in Hoyer’s medium under a stereomicroscope (Leica-EZ4). The slides were dried in an oven (Memmert UN110) at 45 °C for five days. Four to eight females and males were separately mounted from each field and a total of 2512 slides were prepared from the districts of Germencik (356♀, 335♂), Kocarli (276♀, 275♂), Nazilli (298♀, 302♂) and Soke (344♀, 326♂). Female and male slides were morphologically examined using a light microscope (Zeiss Imager A2 DIC). Photomicrographs were captured with an AxioCam 506 color (Carl Zeiss, Germany) digital camera. Pritchard & Baker (1955), Jeppson et al. (1975), Flechtmann & Knihinicki (2002), Seeman & Beard (2011) were used in the morphological identification of Tetranychus species.

Voucher specimens were deposited in the collection of I. Cakmak at the Department of Plant Protection, Aydin Adnan Menderes University, Türkiye.

Molecular verification of Tetranychus urticae and T. turkestani

DNA extraction was performed from the collected populations using the SDS sodium acetate method (Cenis, 1992). For this purpose, ten females from each field were randomly selected under the stereomicroscope. They were transferred to 1.5 ml microcentrifuge tubes and crushed with pestle (Axygen®) for 1 min at 200 rpm in 300 µl lysis buffer (Raeder & Broda, 1985). DNA pellet was dissolved with 25 µl of T10E1 and stored at -20 °C to use molecular studies. The main species found through morphological studies, T. urticae and T. turkestani were subjected to molecular verification using a multiplex PCR method proposed by Sinaie et al. (2018) was used. In PCR studies, multiplex primers UrtF, TurkF and TetCR were used to amplify a 739 bp product for T. urticae and 950 bp for T. turkestani in the ITS region (Table 1). PCR mixture was adjusted to a total volume of 20 μl containing 2 μl of 10 × PCR Buffer (containing 20 mM MgCl2), 0.4 µl dNTP (10 mM each), 0.1 µl DreamTaq DNA Polymerase (Thermo Scientific), 0.08 μl of each primer (UrtF, TurkF and TetCR, 100 µM each), 15.26 µl distilled water and 2 μl of DNA template. PCR was performed using the BioRad C1000 instrument. PCR steps included 30 s of denaturation at 95 °C, 30 s of annealing at 51 °C and 60 s of extension at 72 °C, with a total of 30 cycles. The initial denaturation was performed at 95 °C for 3 min, followed by final extension at 72 °C for 5 min. After amplification, the products were separated in %1.5 agarose gel at 45 V for 45 min. The gel was then stained with GelRed (Biotium) for 30 min and observed under UV transilluminator (302 nm). Bands of about 739 bp were identified as T. urticae and 950 bp as T. turkestani.

Table 1 Primer pairs, approximate product sizes and annealing temperatures used for the identification of spider mites and their endosymbionts
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In sequencing studies, test groups were formed based on the geographical locations where samples were collected, regardless of the sample year. The distances between these groups, including species T. urticae (red and green forms) and T. turkestani, were analyzed using QGIS ver. 3.28, with the Ward’s Lance-William function for the Hierarchical clustering algorithm (QGIS Development Team, 2022). Subsequently, 21 representative populations were selected from these groups. For sequencing of the selected populations, 2 primer pairs specific to both ITS2 (ITS2a, ITS2b) and mtCOI (COI-F, COI-R) gene regions were used for amplification of target regions (Table 1). PCR steps included an initial denaturation at 95 °C for 3 min, 30 s denaturation at 95 °C, 30 s annealing at 55 or 50 °C, 60 s extension at 72 °C for 30 cycles. In addition, it consists of 5 min of final extension steps at 72 °C. The PCR products were sent to Macrogen for sequencing and sequences were determined by Sanger in one-direction using forward primers. The obtained sequences were cropped/checked with 4Peaks (Mac ver. 1.8) software and compared with the sequences of the previously uploaded species in the NCBI gene bank (National Library of Medicine) using a nucleotide blast query. Matching results were identified at the species level by considering the similarity rates and frequency of occurrence (Morgulis et al., 2008; Zhang et al., 2000).

Endosymbiotic bacteria associated with Tetranychus species

The presence of some symbiotic microorganisms such as Wolbachia, Cardinium, Rickettsia, and Spiroplasma species living in mites was investigated using PCR with the primer pairs specified in Table 1. DNA quality and potential PCR defects were accounted for using spider mite generic primers. These studies relied on genomic DNA extracts as the basis for identifying mites.

Populations were grouped according to T. urticae (red and green forms) and T. turkestani, based on their geographical locations and distances using QGIS ver. 3.28 (QGIS Development Team, 2022). A total of 21 samples from studied mite sequencing were also included. Using these groups, endosymbiont identification studies were carried out on 70 separate populations. Amplification was performed by qPCR (BioRad CFX96) with a total of 10 µl mix (SsoFast EvaGreen Supermix, BioRad). qPCR steps consisted of the following: a 3-min initial denaturation at 95 °C, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 52–65 °C (see Table 1) for 30 s. A final step with a temperature sequence for melting curve analysis was included. Data readings were performed at the end of each extension step from the FAM channel. The study also incorporated control; in Tetranychus evansi (teva, 6M1), Tetranychus ludeni (tlu), Tetranychus urticae (green form, turtg, Santport2), Tetranychus urticae (red form, turtr, SB9) were obtained from Dr. Flore Zélé (University of Montpellier, France) and Eutetranychus orientalis (eu) were used as positive and negative controls. Cq values were evaluated using BioRad Maestro (ver. 5.2.008.022) program.

Results

Morphological identification of Tetranychus species and their prevalence

Spider mites were found in 196 out of 220 cotton fields that were visited in Germencik, Kocarli, Nazilli and Soke districts of Aydin province in 2020 and 2021. Based on the slides of female and male samples, two spider mite species, Tetranychus urticae (red form [RF] and green form [GF]) and T. turkestani, were identified (Fig. 2).

Fig. 2
figure 2

Aedeagus shapes of the species identified as Tetranychus turkestani A and T. urticae B

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Tetranychus urticae is close to T. turkestani by male: dorsal margin of knob of aedeagus angulate flat or evenly rounded; empodia I–II each with an obvious dorsal spur, empodium I clawlike (uncinate), empodia II–IV with proximoventral hairs long and free; peritreme hook. Female: tarsus I with sockets of 4 tactile setae proximal to the socket of the proximal duplex setae; dorsal striae longitudinal between setae e1–e1 and f1–f1 forming a diamond-shaped pattern between setae e1 and f1; dorsal striae with lobes, but it can be distinguished by its different aedeagus (knob of aedeagus small, less than twice as long [about 1.5 ×] as the width of the neck; dorsal margin of knob tends to be angulate in T. urticae vs. knob of aedeagus with large posterior projection, its length approximately equal to the width of the neck; dorsal margin of the knob angulate in T. turkestani) (for more information, see Zhang, 2003).

Tetranychus urticae was detected at a rate of 60.7% (57.1% red form and 3.6% green form) in 119 out of 196 cotton fields. In 77 fields, T. turkestani was found at a rate of 39.3%. T. urticae RF was extensively found in Germencik (89.2%) and Soke (87.8%), whereas T. turkestani was found in Kocarli (65.9%) and Nazilli (90.2%) in 2020–2021. T. urticae RF was detected only in Germencik and Soke, while T. urticae GF was solely found in Nazilli. However, both T. urticae RF and GF were found in Kocarli (Table 2).

Table 2 Spider mite species and their occurence frequency (no. of fields/rates) obtained from cotton fields in Germencik, Kocarli, Nazilli and Soke districts of Aydin province in 2020–2021
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Molecular verification of Tetranychus urticae and T. turkestani

Multiplex PCR studies conducted with UrtF, TurkF, and TetCR primers revealed that out of the 196 populations examined, 119 were identified as T. urticae (112 red, 7 green form), and 77 were determined to be T. turkestani. Gel electrophoresis photographs of 6 representative populations selected from all samples are given in Fig. 3. Molecular test results were found to be compatible with morphological identification results.

Fig. 3
figure 3

Agarose gel electrophoresis of PCR products amplified using UrtF, TurkF and TetCR primers (Lanes 2–4: Tetranychus urticae [N6G20, S5R20, G23R21], Lanes 5–7: Tetranychus turkestani [G7G20, S18G21, K2G21], Lane 1: GSS population as compare control, Lane 8: Distilled water as negative control, Lane M, Molecular marker 500–10000 bp [Fermentas SM1128])

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In sequence analyses, ITS2 gene region enabled classification of 6 out of the 21 collected populations as T. urticae (identity > 99.36%) and 11 populations as T. turkestani (identity > 99.5%). The remaining four populations were discarded because of poor sequence quality for this gene region. In COI sequences, between 384 and 407 bp sequence were obtained and matched 9 out of 21 individuals as T. urticae (identity > 99.49%) and 10 populations as T. turkestani (identity > 99.75%). The sequences of remaining two populations were discarded because of poor sequence quality. While both ITS2 and COI gene sequence were obtained from fifteen samples, sequences of ITS or COI were obtained from six samples. Thus, 21 of the sequenced populations were identified at the species level in parallel with the results of the multiplex PCR test and morphological identification.

Endosymbiotic bacteria associated with Tetranychus species

The qPCR analysis was performed with spider mite primers (ITS1G-F and ITS1G-R) in all 70 spider mite populations used for endosymbiont detection. The results confirmed that negative reactions can be distinguished from PCR defects by obtaining a Cq value between 17.13 and 35.52 (Threshold: 207.59; major single peak at 79 °C in Melting curve analysis). The qPCR analysis showed that twenty-nine populations were infected with only Wolbachia (with wsp81F and wsp691R primers, Cq values: 26.48–35.64, major single peak at 80 °C in melting curve). Tetranychus turkestani populations showed higher infection rates (21) compared to T. urticae (8). Fourteen populations were infected with Rickettsia (with RICS741F and RCIT1197R primers, Cq values: 23.73–35.35, major single peak at 87 °C in melting curve). Higher rates were found in T. urticae (10) compared to T. turkestani (4). One population each of T. urticae and T. turkestani was infected with both Wolbachia and Rickettsia (Table 3). No Cardinium or Spiroplasma was detected in the populations (while positive controls previously known to contain this symbiont for Cardinium gave positive results as expected, a positive control for Spiroplasma could not be obtained, so the populations were evaluated as probable negative). In each endosymbiont study; although an appropriate Cq value was obtained, samples showing a different melting temperature than the positive controls were considered as non-specific reaction and were considered negative for a specific endosymbiont.

Table 3 The number of endosymbionts in spider mite populations
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Discussion

Field sampling conducted in Aydin province over a two- year period, revealed that 89% of cotton fields (196 out of 220) were infested with spider mites. Additionally, our results showed Tetranychus urticae RF (57.1%) and T. turkestani (39.3%) as the most prevalent species, with a lesser incidence of T. urticae GF (3.6%). This aligns with previous research in Türkiye which found that T. urticae, T. cinnabarinus, T. turkestani and T. desertorum were common spider mite species in cotton fields (Düzgüneş, 1962; Dinçer, 1975). We agree that T. cinnabarinus is a junior synonym of T. urticae (Auger et al., 2013). Tetranychus desertorum, which was detected once in 1962, was not found in this study or in recent literature. This suggests that it may be rare or that there may have been a misidentification (Migeon & Dorkeld, 2024).

Tetranychus urticae and T. turkestani are globally distributed mite species that cause damage and yield loss to a variety of vegetable, field and ornamental crops. This study demonstrated the exclusive presence of T. urticae and T. turkestani in cotton fields, which is consistent with findings by Ben-David et al. (2007) in agricultural habitats in Israel. However, the coexistence of these species was later reported in these fields (Ben-David et al., 2009). It is possible that they may co-occur in Aydin cotton fields in future. Similar multi-species infestations have been documented in other parts of the world. Tetranychus turkestani, T. urticae and T. pacificus McGregor were identified as the primary tetranychid mite species responsible for cotton damage in the San Joaquin Valley in California (Carey & Bradley, 1982). All three species were present in all areas where cotton was cultivated. Guo et al. (2013) reported the coexistence of T. turkestani and T. truncatus in cotton fields in Xinjiang, with T. turkestani potentially displacing the dominant T. truncatus in some areas.

This study is the first report on the occurrence and the prevalence of T. urticae RF & GF and T. turkestani in the cotton fields of Türkiye. Notably, T. urticae RF & GF was detected at a significantly higher rate (60.7%) compared to T. turkestani (39.3%). The analysis of species incidence in terms of locations and years showed that T. urticae RF dominance in Germencik and Soke (25 km and 15 km from the coast, respectively), whereas T. turkestani was more common in Nazilli (110 km far from the sea). Despite being prevalent in Kocarli (60 km from the sea), both T. urticae RF and GF were also detected alongside T. turkestani. These findings are in alignment with ecological requirements of the species. T. urticae GF thrives in cold temperate latitudes with hot and dry summers, while T. urticae RF prefers warmer temperate and subtropical regions, or greenhouses in northern geographical regions (Auger et al., 2013). During the cotton growing season, the average temperature in areas dominated by T. turkestani (Nazilli) was similar to areas where T. urticae RF (Soke) was detected, ranging between 25–35˚C. However, Soke had higher relative humidity (50–75%) compared to Nazilli (45–55%) (Meteoblue, 2024a, 2024b). High temperatures and low rainfall are more favorable for T. turkestani development (Jeppson et al., 1975), supporting the results of this study. Conversely, Carey and Bradley (1982) reported that T. urticae was found mainly in the eastern and southern regions of the San Jaquen Valley, while no distinct regional pattern was observed for T. turkestani in cotton. These areas typically have the Mediterranean climate, with rainy winters and dry summers. Simons (1964) noted distinct colonization patterns. Tetranychus turkestani forms dense aggregations, while T. urticae is more dispersed. This aligns with the greater damage potential of T. turkestani observed by Jeppson et al. (1975). The present study suggests this species shift might explain the recent increase in Tetranychus-related damage within the region.

In our study, we found Wolbachia in 9 out of 36 T. urticae populations and 22 of the 34 T. turkestani populations, whereas Rickettsia was found in 11 out of 36 T. urticae populations and 5 out of the 34 T. turkestani populations. Cardinium and Spiroplasma were not found in any of the sampled mites. These endosymbiont genera are common in arthropods (Jeyaprakash & Hoy, 2000). Since these endosymbionts may facilitate pesticide resistance, further research is required to investigate the specific relationships between the observed endosymbionts and Tetranychus species in sampled cotton fields. This may clarify the development of resistance, either at the species level or through endosymbiont interactions.

Ongoing research conducted in the province of Aydin has demonstrated a correlation between the intensity of acaricide usage and the dominant Tetranychus mite species. Areas with a high prevalence of T. urticae RF populations had more frequent spraying compared to regions with T. turkestani predominance (unpublished data). This observation is supported by studies that indicate T. urticae GF develops resistance to acaricides at a higher rate compared to other species (Lu et al., 2016), which may explain the increased use of spraying. However, studies have also shown a significant variability in acaricide resistance even within T. urticae RF populations (Yalçın et al., 2018). Specific populations can have different levels of resistance depending on the chemical compound, which could potentially complicate pest management strategies. The observed discrepancy in acaricide usage within Aydin province may be attributed to the susceptibility of T. turkestani to common acaricides, in contrast to the varying levels of resistance found in T. urticae. Historical studies further corroborate this notion (Grafton-Cardwell et al., 1987; Bruce-Oliver & Grafton-Cardwell, 1996; Shen et al., 2017; Sohrabi & Ziaee, 2021).

In conclusion, our findings indicate the prevalence of tetranychid mites in Aydin cotton cultivation with T. urticae RF and T. turkestani as the most prevalent species. Areas with T. urticae RF were reported to have a higher acaricide usage. This could be related to acaricide resistance development within this population. Conducting additional research to investigate the distribution and the resistance levels of these tetranychid mite species, and the potential overlap of niches in cotton-growing areas is of utmost importance. This vital information will contribute to the development of sustainable spider mite management practices by enabling us to predict and anticipate shifts in species composition in the future.