Stethorini (Coleoptera: Coccinellidae: Coccinellinae) of South India: their associated mite species and barcode gap analysis

Abstract

Background

Stethorini are specialized mite predators in the family Coccinellidae, which are small with black body and brown or yellow colored appendages. This study aimed to survey species of Stethorini associated with phytophagous mites of South India and their prey range.

Results

Eight species of Stethorini, viz., Parastethorus sp. Stethorus (Allostethorus) forficatus, S. (A.) pauperculus, S. (A.) tetranychi, S. (A.) sp., S. keralicus, S. (S.) rani and S. (S.) sp., each associated with distinct species of mite were identified in this study and part of mitochondrial cytochrome c oxidase subunit I sequences of seven of these species are provided. Barcode gap analysis and distance summary analysis in the BOLD system as well as phylogenetic analysis of these seven species were given.

Conclusions

This study revealed the species of Stethorini in South India along with their prey range and associated plants. As all the species look similar with black body and yellow appendages, DNA barcodes generated can be used as an effective bio-identification tool for this group.

Background

Stethorini sensu stricto Dobzhansky is a group of coccinellid predators that are highly specialized feeders on mites belonging to the family Tetranychidae (spider mite) and Tenuipalpidae (false spider mite or flat mite). They pierce the prey, siphon prey juices and regurgitate them back and finally imbibe the juices, leaving the exoskeleton (Houck 1991). Stethorini occur in diverse habitats and many feed on different prey species, while some are more specialized, preferring to feed exclusively on certain species. Species of Stethorini have been used as potential biological control agents of spider mites and many species have been intentionally introduced throughout the world (Biddinger et al. 2009).

Members of Stethorini are grouped into two genera, viz., Stethorus Weise and Parastethorus Pang & Mao under the tribe Coccidulini of subfamily Coccinellinae (Seago et al. 2011). Earlier, Dobzhansky (1924) erected Stethorini as a tribe under the subfamily Scymninae to place a single genus Stethorus, which was earlier included under the tribe Scymnini. Kapur (1948) stated that the generic nomen Stethorus was originally proposed by Weise in 1885 as a subgenus of Scymnus and was later elevated to generic status by Weise & Casey in 1899. Stethorus was further divided into three subgenera, viz., Allostethorus Iablokoff-Khnzorian, Parastethorus and Stethorus based on the characters of genitalia and later, Slipinski (2007) raised subgenus Parastethorus to generic status. Based on morphology and a multilocus molecular dataset, Seago et al. (2011) revised the sub-familial classification of the family Coccinellidae and included the tribe Stethorini as part of the tribe Coccidulini. Members of Stethorini are cosmopolitan and the world fauna consists of more than 100 species (Biddinger et al. 2009) of which ten species are reported from India (Poorani 2002).

Adults of all species of Stethorini are small (< 2 mm), pubescent, black with brown or yellow colored antennae, mouth parts and legs. As these are cryptic in nature, misidentifications are commonly reported and therefore this group was a candidate for further diagnostic taxonomy using molecular barcoding methods (Biddinger et al. 2009).

Mitochondrial gene cytochrome c oxidase subunit I (COI) has been used widely as a ‘barcode’ for bio-identification in animals (Hebert et al. 2003). This has been used as a tool for the identification of many insects including beetles (Zhao et al. 2017). Huang et al. (2020) used mini barcoding primers to generate DNA barcode of 104 coccinellid species.

The objectives of this study were to identify species of Stethorini associated with phytophagous mites of South India, understand their prey range and provide barcodes of the species in order to facilitate identification of this otherwise cryptic group of beetles.

Methods

Sample collection and preservation

Extensive surveys were carried out in 27 locations covering three South Indian states, viz., Kerala, Karnataka and Tamil Nadu during 2015–2017 for the collection of Stethorini associated with phytophagous mites. Field crops, plantation crops, vegetables, fruits, spices, ornamentals and weeds were examined and the beetles were collected using aspirators. The grubs and pupae were also collected during the survey and were reared to adults. The beetles were preserved in 99% alcohol at − 80 °C for DNA extraction. For morphological characterization and identification of species, the beetles were either dry preserved by mounting on paper points or wet preserved in 70% alcohol. During the survey, the associated prey mites were also collected along with the infested leaf samples in polythene bags. In the laboratory, slides were prepared by mounting adult female and male mites separately on dorsal position in Hoyer’s medium on glass slide. Morphological characters, viz., chaetotaxy of hysterosoma and legs, structure of empodium were used for genus level identification. Males of spider mites were also mounted in lateral position (Henderson 2001) for observing the shape of the aedeagus to determine the species.

Identification of Stethorini and prey mites

Species of Stethorini were identified based on available literature and taxonomic keys (Kapur 1948, 1961 and Poorani 2017). Mite specimens were identified based on available species descriptions and taxonomic keys provided by Gupta (1985), Gupta and Gupta (1994), Ehara (1995), Srinivasa et al. (2012) and Zeity et al. (2016).

DNA extraction, PCR amplification

DNA was extracted using Qiagen DNeasy® blood and tissue kit following manufacturer’s protocol. The quantity and quality of DNA samples were assessed using Nanodrop spectrophotometer by measuring the absorbance at wavelength of 260 nm and 280 nm (JenwayGenova Nano). The AE buffer in the extraction kit, in which the isolated DNA was stored, was used as blank to measure the absorbance.

The DNA extracts were subjected to polymerase chain reaction (PCR) in order to amplify 658 bp region near the 5′ terminus of the mitochondrial protein coding gene, mitochondrial cytochrome c oxidase subunit I, by following standard protocols. The reaction was carried out in 20 μl reaction volume containing 10 μl EmeraldAmp® GT PCR Master Mix, 9 μl of molecular water + template DNA and 0.5 μl each of forward primer and reverse primer. The forward and reverse primers used were LCO 1490: 5′-GGTCAACAAATCATAAAGATATTGG-3′ and HCO 2198: 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ respectively (Folmer et al. 1994). The PCR was performed using Eppendorf Master Cycler gradient thermal cycler. Thermal cycling included an initial denaturation of 94 °C for 4 min, followed by 30 cycles of denaturation of 94 °C for 30 s, annealing 60.1 °C for 1 min and extension of 72 °C for 2 min, followed by final extension of 72 °C for 10 min. The amplified product was analyzed by Agarose Gel Electrophoresis (AGE) in 1.2 per cent agarose gel.

Sequencing and data analysis

PCR products were bidirectionally sequenced using Sanger technology at AgriGenome Labs Pvt Ltd, Kochi, Kerala, India. Sequencing was done by using Big Dye 3.1 cycle sequencing kit and the instrument used was sequencing machine ABI 3730 XL DNA Analyzer. The forward and reverse sequences were aligned to check for nucleotide mismatches at specific positions. The final alignment was assembled in CAP 3 and was checked for stop codons using MEGA7 (Molecular Evolutionary Genetics Analysis) (Kumar et al. 2016). Homology search with the available database was done using NCBI BLAST. The sequences generated were submitted to GenBank (NCBI) and BOLD (Barcode of Life Data Systems).

BOLD analysis

Seventeen sequences of seven species of Stethorini were subjected to barcode gap analysis and distance summary analysis in BOLD. Barcode gap analysis provides the distribution of distance within species and distance to the nearest neighbor. The distance summary gives the distribution of within species divergence to between species divergence for the samples used in the study. The distance model used in both analysis was Kimura 2 Parameter. K2P was used as the model as it has been used widely in DNA barcoding literature and hence it facilitates comparisons with earlier works (Candek and Kuntner 2014). BOLD aligner (Amino Acid-based HMM) was used for the alignment of sequences.

Phylogenetic analysis

Fifteen COI sequences from India comprising 6 putative species of Stethorus and three from one species of Parastethorus were generated as part of this study. Nine sequences of Stethorus were obtained from NCBI that belong to the species S. punctillum Weise, S. pusillus (Herbst) and S. punctum (LeConte). Representatives of the genus Scymus (4 sequences) were used as out-group according to the phylogeny published by Giorgi et al. (2009). The sequences used for phylogenetic analyses are listed in Table 1.

Table 1 Sequences used for phylogenetic analysis

The maximum likelihood (ML) algorithm was used for phylogenetic inference as implemented in the online version of IQ-TREE (Trifinopoulos et al. 2016), where branch support was evaluated using ultrafast bootstraps (Hoang et al. 2018). The sequence type was set to Invertebrate mitochondrial (CODON 5 in IQ-TREE) and data were partitioned using the edge linked method by codon position. The substitution models for each partition recommended and used by IQ-TREE are listed in Table 2. Ultrafast bootstrapping and single branch tests were performed using 1000 replicates (alignments) and a correlation coefficient of 0.99. Parameters for tree search were left at default values.

Table 2 Substitution model recommended for each partition

Results

Species of Stethorini and their prey range

Eight distinct lineages of Stethorini belonging to two genera, viz., Parastethorus and Stethorus associated with seven species of mites of families Tetranychidae and Tenuipalpidae were identified in this study. Genus Stethorus was represented by two subgenera, Allostethorus and Stethorus. Of the eight lineages, five were identified as nominal species, two up to the subgenus and one at the generic level. Species identified included Stethorus (Allostethorus) forficatus Poorani, S. (A.) pauperculus (Weise), S. (A.) tetranychi Kapur, S. keralicus Kapur and S. (Stethorus) rani Kapur. The prey and associated host plants of Stethorini observed during this study are given in Table 3.

Table 3 Stethorini species associated with different mite species

Sequence analysis and DNA barcoding

Eighteen sequences were generated for seven species and homology analysis of these sequences in BLAST showed a similarity that ranged from 88 to 84% with the sequences in NCBI database, except for Parastethorus which showed 95 per cent similarity with a species of coccinellid, whose species identity is not mentioned. Generated sequences were submitted in GenBank and BOLD and the details of NCBI accession number and Barcode index number are given (Table 4).

Table 4 NCBI accession number and Barcode index numbers for species of Stethorini

BOLD analysis

The distribution of sequence divergence at each taxonomic level using distance summary analysis and mean intraspecific distance, maximum intraspecific and distance to nearest neighbor using Barcode gap analysis in BOLD are given (Tables 5 and 6). The mean distance within species was 0.68%, whereas that of within genus and within family was found to be 19.10 and 19.34%, respectively. The minimum and maximum divergences within species were recorded as zero and 2.73. The maximum intraspecific distance varied from 0 to 2.73, whereas the distance to the nearest neighbor varied from 13.67 to 19.27.

Table 5 Distribution of sequence divergence

Table 6 Comparison of species with nearest-neighbor species

Phylogenetic inference

The phylogenetic reconstruction presents a strong support for all branches leading to species-level lineages, but not for branches distending from deeper nodes in the tree (Fig. 1).

Fig. 1

A maximum-likelihood-based phylogenetic reconstruction of the Indian species of Stethorus and Parastethorus. The numbers represent bootstrap support for the respective branches as evaluated based on the online version of IQ-TREE

Nevertheless, the monophyly of Stethorus + Parastethorus was strongly supported (BS 100). However, the composition of the two broad clades within it and the phylogenetic position of Parastethorus cannot be inferred due to the lack of robust branch support. The monophyly of all species-level lineages is recovered with strong support (BS 98–100), reinforcing the taxonomic validity of the species from a phylogenetic perspective. Phylogenetic inference suggests that the sequence of S. pusillus was nested within the S. punctillum clade. It was understood that S. pusillus and S. punctillum were synonyms.

Discussion

Species of Stethorini and their prey range

Stethorini consists of two genera, viz., Parastethorus and Stethorus. The present study included one species of Parastethorus and seven species of Stethorus. There are 14 species of Parastethorus reported around the world (Li et al. 2015). Parastethorus indira (Kapur) is the only species of Parastethorus reported from India so far. Genus Stethorus is represented by the subgenera Allostethorus and Stethorus. World fauna of Stethorus consists of approximately 94 species, of which 38 belong to Stethorus (Allostethorus). Ten species belong to two genera, viz., Parastethorus indira, S. gilvifrons (Mulsant), S. keralicus, S. parcepunctatus (Kapur), S. pauperculus, S. rani, S. tetranychi (Poorani 2002), S. forficatus (Poorani 2017), S. aptus (Govindasamy and Khursheed 2018) and S. gangliiformis Li, Chen & Ren (Janakiraman and Thangjam 2019) were reported from India so far.

Stethorini is the only mite specialist predator under the family Coccinellidae (Biddinger et al. 2009) feeding on tetranychid or tenuipalpid mites. Stethorini usually feed on different species of tetranychid mites, but a few are specialised on some species. For instance, S. punctillum and S. gilvifrons do not prefer to feed on or reproduce on the tetranychid mites of the genus Bryobia (Kaylani 1967). There are reports on the utilization of non-tetranychid or non-tenuipalpid mite as prey, especially during starvation (Biddinger et al. 2009).

Prey ranges of different species of coccinellids reported from India were catalogued by Pervez (2004), in which they also mentioned non-mites as prey of Stethorus. Biddinger et al. (2009) opined that reports of Stethorini feeding on non-acarid prey and non-prey foods challenge the argument that Stethorini are specialized mite predators. However, they believed that superficial similarity of Stethorus to some species of Delphastus, Scymnus and Telsimia might have accounted for most reports of Stethorini feeding on non-mites like scale or aphids. During the present study, we could observe Stethorini feeding only on tetranychid and tenuipalpid mites. In this study, S. (A.) pauperculus recorded a wider host range included Oligonychus indicus, Tetranychus macfarlanei, T. truncatus and T. okinawanus. Stethorus keralicus was found to be more specialized and recorded only on a tenuipalpid mite, Raoiella indica. Carrillo et al. (2012) considered S. keralicus specific to the mites of the genus Raoiella. Interestingly, whenever an infestation of R. indica was observed on arecanut, S. keralicus was found in association with the mites. Daniel (1981) considered S. keralicus as the most important predator of R. indica in Kerala. Stethorus keralicus was a voracious predator of all stages of R. indica and was found throughout the year (Puttaswamy and Rangaswamy 1976).

DNA barcoding

Among the 17 sequences (seven species under two genera) generated during the present study, there was no overlap between intraspecific and interspecific variations. In this dataset, the maximum intraspecific distance was relatively low (0–2.73) and the nearest-neighbor distance between species was found to be higher (13.67–19.27). This may be due to the smaller number of samples in the study. There was a significant positive correlation between the number of specimens sampled for a species and its maximum intraspecific divergence (Pentinsaari et al. 2014). However, among the species studied the maximum intraspecific distance was lower than the minimum interspecific distance.

The analysis showed that the nearest-neighbor species of Parastethorus sp. was S. (A.) pauperculus, S. (A.) sp. was S. (A.) forficatus, and S. (S.) sp. was Stethorus (Stethorus) rani. Meier et al. (2008) suggested that in order to check the barcode gap the overlap between the lowest interspecific and highest intraspecific genetic distance has to be considered. They estimated that for Coleoptera, the mean intraspecific variability (%), mean interspecific variability (%) and smallest interspecific variability (%) were 2.0 ± 2.3, 11.2 ± 4.3 and 7 ± 5.4, respectively. The study of North European beetle fauna by Pentinsaari et al. (2014) showed that the nearest-neighbor divergences among species of Coleoptera were higher than those detected in other groups such as Lepidoptera and the overall average K2P divergence between nearest-neighbor species for Coleoptera was 11.99%. They also reported that for Coccinellidae the minimum, mean and maximum divergences within species were (zero, 1.34 and 7.83), respectively. The minimum, mean and maximum distances to nearest-neighbor species were estimated as (zero, 11.91 and 21.12), respectively.

In the present study, it was observed that maximum interspecific distance was more than maximum intergeneric distance. This means species within a particular genus was found to be more divergent than species within two different genera. This is more evident when the species were compared with nearest-neighbor species. Genetic distance of S. keralicus to its nearest neighbor (S. (Allostethorus) sp.—19.27) was larger than the genetic distance of Parastethorus sp. to its nearest neighbor (S. pauperculus—16.62). Among the species studied, S. keralicus was the most genetically distant species. Stethorus keralicus was found to be distinct from other species of Stethorini with 10 antennomeres and variation in shape of male genitalia and spermatheca. Hence, the question arises whether S. keralicus has to be treated separately, for which further studies are needed.

Phylogenetic analysis

The position of Parastethorus in the phylogeny presented here causes paraphyly within Stethorus. In order to maintain cladistic correctness, i.e., the monophyly of Stethorus, Parastethorus must be subsumed under the former. However, this phyletic relationship must be tested further using nuclear DNA to be able to make a tangible inference, and therefore, we refrain from making any taxonomic changes at this stage. Given the morphological similarities between Stethorus and Parastethorus, we posit that the latter might just be a divergent lineage of the broader Stethorus radiation. Similarly, the species S. keralicus was sister related by a well-supported (BS 100) long branch to a broad clade comprising S. rani, S. punctum, S. punctillum and an unidentified species. Given its morphological dissimilarities with other Stethorus sp., this relationship was expected to change with better sampling. Stethorus were poorly sampled for genetic studies from across the world, with very few sequences publicly available for use. Further phylogenetic studies that incorporate near-complete sampling in terms of species and a larger genetic dataset will be able to resolve relationships within this cosmopolitan group of beetles.

Conclusion

Eight species of Stethorini belonging to two genera were identified along with their prey and associated host plants from South India. The sequences of seven species of Stethorini were submitted to NCBI and BOLD for the first time. Cryptic characters of adults of Stethorus might have led to species misidentification in the literature. As all the species look similar with black body and yellow appendages, sequences of COI will be used for easy and reliable identification. Thus, DNA barcoding and estimation of genetic distance can be used as an effective tool in separating the species and hence complementary to conventional taxonomy.