Telenomus nizwaensis (Hymenoptera: Scelionidae), an important egg parasitoid of the pomegranate butterfly Deudorix livia Klug (Lepidoptera: Lycaenidae) in Oman.

The pomegranate butterfly Deudorix (= Virachola) livia is the major pest of pomegranate, a crop of economic importance, in Oman. A species of parasitoid wasp in the hymenopteran family Scelionidae is responsible for high levels of mortality of its eggs. This wasp is described herein as Telenomus nizwaensis Polaszek sp. n., based on morphology and DNA sequence data. T. nizwaensis is currently known only from D. livia, which is also a pest of economic importance on other crops in North Africa, the Arabian Peninsula, and the Mediterranean. We summarise current knowledge of T. nizwaensis life-history and its potential to provide biological pest control.

Introduction

Pomegranate Punica granatum L. (Lythraceae, Punicoideae) is a fruit-bearing deciduous shrub or small tree that originates from the Near-East, where it has been cultivated for centuries [1]. It is increasingly important in other parts of the world, such as China, SE Asia, and the Americas [2-4]. In Oman, pomegranate is grown mainly in the mountainous region of Al Jabal Al Akhdar (near the city of Nizwa, Ad Dakhiliyah Governorate), with a total of more than 22,000 productive trees [5]. They are grown at ca. 3000m, where the temperature averages 24°C during the summer fruiting season [6]. Mature fruits are both sweet and large, and in Oman can sell for the equivalent of US$5 per fruit in some seasons. Hence, pomegranate cultivation is a major aspect of the agricultural economy [7]. Unfortunately, during each growing season Omani pomegranates are infested by a lepidopteran pest called the pomegranate butterfly, Deudorix (= Virachola) livia Klug (Lepidoptera: Lycaenidae) [8], and this causes damage of economic importance. The larval stage of D. livia burrows within the fruit, causing spoilage of seeds, subsequent infection by saprophytic bacteria and growth of moulds, rotting of part or all of the fruit, and, in some cases, the fall of the fruit.

Attempts to control D. livia in Oman include an annual IPM program using physical (fruit bagging), chemical control (1–2 applications of Deltamethrin during high infestation periods) and mainly biological control using parasitoid wasps. Trichogramma species (Chalcidoidea: Trichogrammatidae) have been imported for release, and a species of Telenomus (Platygastroidea: Scelionidae) is naturally present in the Al Jabal Al Akhdar mountains. Specimens of these Telenomus were sent to the first author (AP) in 2008, and examined morphologically, including the preparation of male genitalia, critical for recognising Telenomus at species-level. The conclusion based on morphology alone was that these specimens belong to an undescribed species. Because of the difficulty in unequivocally establishing them as a species new to science on the basis of morphology alone, they were not formally described at that time. With the advent of relatively rapid and affordable techniques of DNA extraction and sequencing, it recently became feasible to present a formal description, using an integrated taxonomic approach. Specimens from the same host and locality as the initial sample were sent to the first author in 2018. These underwent a DNA extraction protocol that leaves the sclerotized structures intact, and the resulting DNA sequence data supported the initial conclusion that the specimens belonged to an undescribed species. Here we describe this new species as Telenomus nizwaensis Polaszek, both to facilitate future identification, and to provide the formal nomenclature essential to support further work using this parasitoid as a biological control agent. The name of this new species is derived from Nizwa, the ancient capital of Northern Oman, about 60 km from Al Jabal Al Akhdar. Further, we provide a summary of its currently known biology and areas for further research.

Materials and methods

Specimen repositories: Abbreviations

Individual specimen numbers are given below under “Material examined”. Specimens are permanently publicly deposited in the following repositories:

NHMUK: Natural History Museum, London UK.

ONHM: Oman Natural History Museum, Muscat, Oman.

USNM: National Museum of Natural History, Smithsonian Institution, Washington D.C., USA.

Morphological study

Specimens were obtained from the field by rearing from egg masses of D. livia (R.A. and A.A., unpublished data). Because the specimens were collected in Oman by Omani researchers from the Ministry of Agriculture, Fisheries and Water Resources, no collecting permits were required for the described study. During the pomegranate fruiting season (April-September), eggs were collected from fruits, leaves and occasionally from stems. They were placed in Eppendorf tubes and transferred to the laboratory in a cool box (to avoid desiccation). In the laboratory, eggs were kept at a maximum of 5 eggs per tube to limit crowding. When adult parasitoids emerged, around three weeks later, they were stored in a new tube in 100% ethanol. Card-mounted specimens were observed with a Leitz binocular microscope at magnifications ranging from 10× to 40×. Slide-mounted structures were observed with a Leitz Dialux 20 EB compound microscope at magnifications ranging between 40× and 400×. Several specimens were gold-palladium coated and photographed with a Zeiss Ultra Plus field emission Scanning Electron Microscope at magnifications between 300× and 900×. Images were generated as follows: Light microscope images: Canon DSLR with 10× Mitutoyo objective, processed with HeliconFocus stacking software; Compound microscope images (slide-mounted structures): Leitz Dialux 20EB compound microscope using Nomarski Differential Interference Contrast (DIC) illumination, photographed with MicroPublisher 5.0 RTV camera; scanned sections stacked and combined using Synoptics AutoMontage® software; Scanning electron micrographs: Zeiss Ultra Plus field emission Scanning Electron Microscope. All final image editing was with Adobe Photoshop CC®. Morphological terminology largely follows Johnson [9] and Talamas et al. [10]; genitalia terminology follows Johnson [9] and Polaszek and Kimani [11]. The holotype of T. nizwaensis is deposited in NHMUK, paratypes in NHMUK, ONHM and USNM.

DNA sequencing

Eight Telenomus nizwaensis individuals from Oman (4 females, 4 males) were subjected to “non-destructive” DNA extraction. Genomic DNA was extracted using established protocols [12, 13], which leave the sclerotized parts of the specimen intact. Specimens were then critical point dried and card-mounted, with selected individuals then dissected and mounted in Canada balsam on microscope slides, and others gold-palladium coated for SEM examination.

To generate CO1 sequences, the standard “barcode” forward primer LCO1490 [14] was paired with the reverse primer C1-N-2329 [15], resulting in an amplicon that is longer than the standard “barcode” region by about 160 bp. The PCR cycle for the 5′ end of the CO1 consisted of an initial denaturation step of 94°C for 2 min, followed by 40 cycles of 94°C for 30 s, 40°C for 60 s and 72°C for 30 s, and a final extension step of 10 min at 72°C. For 28S the conditions where similar except for annealing at 55°C for 30 s.

The 28S D2 fragment was amplified with the primers D23F (5′-GAG AGT TCA AGA GTA CGT G-3′) [16] and D2R (aka 28S-Rev) (5′-TTG GTC CGT GTT TCA AGA CGG-3′) [17]. All reactions were carried out in a 25μl reaction volume containing 5μl of template DNA, 2.5μl of 10× PCR buffer, 0.75μl of 50 mM MgCl2, 0.2μl dNTPs solution (25 mM each), 1.25μl of each primer (10μM), 0.3μl Taq polymerase (5u/μl Biotaq, Bioline) and PCR grade water to final volume.

Both DNA strands were sequenced at the Natural History Museum Life Sciences DNA Sequencing Facility (London) using the same primers used for the PCR. Forward and reverse sequences were assembled and edited in Sequencher version 4.8. Contigs of the 28S rDNA and COI genes were then assembled using GeneStudio Professional Edition (v. 2.2.0.0).

Phylogenetic analyses

Phylogenetic analyses were conducted using the CO1 and 28S sequences of three T. nizwaensis specimens (specimens subsequently designated male holotype and 2 male paratypes) and additional platygastroid sequences retrieved from GenBank. Sequence alignments of both genes were generated with MAFFT (v. 7.394) [18] using the L-INS-i algorithm.

Phylogenies were estimated for 28S, COI, and 28S+COI in IQ-TREE (v. 1.6.12) [19]. The best nucleotide substitution model for each gene was selected with ModelFinder [20]. The combined analysis (28S+CO1) was run with two partitions, one for each gene, using the best nucleotide substitution model identified in the previous step [21]. Branch support was estimated with 1000 ultrafast bootstrap replicates [22]. The trees presented are those with the best log-likelihood score from 100 independent runs. Trissolcus thyantae Ashmead (Scelionidae: Telenominae) was selected as the outgroup in each analysis based on the results of Taekul et al. [23].

Nomenclatural acts

The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:CE45C3B6-038B-4-9EA5BA8254BCORRECT. The electronic edition of this work was published in a journal with an ISSN and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Description

urn:lsid:zoobank.org:act:148B35EB-4B8B-44E4-AC56-1F4BC5F7050CORRECT

Figs Male (holotype): length 0.75 mm.

Scanning electron micrograph of terminal antennomeres showing papillary sensilla present on A8-11 and clearly absent from A7.

Colour (holotype): almost entirely dark brown-black, with the following paler (Figs 1 and 2): apices of all tibiae; basitarsi. Wings hyaline.

Fig 1

Telenomus nizwaensis Polaszek male holotype.

Lateral habitus.

Fig 2

Telenomus nizwaensis Polaszek male holotype.

Dorsal habitus.

Morphology (holotype): Vertex smoothly rounded onto occiput; entire vertex with deep reticulate sculpture (Fig 3); occiput entirely smooth, occipital carina present, higher and weaker centrally (Fig 4); hyperoccipital carina absent; frons smooth, reticulate sculpture present between lower inner eye margins and toruli; interantennal process and frontal depression absent. Frons moderately convex between inner orbits and toruli; eyes setose; malar region smooth; malar sulcus present; gena with reticulate sculpture behind eyes. Antennae 11-merous.

Fig 3

Telenomus nizwaensis Polaszek male paratype.

Face.

Fig 4

Mesoscutum flattened, entirely reticulately sculptured except posterior lateral corners (above axillae) which are smooth; scutellum mostly smooth; axillae smooth. Lateral portion of scutoscutellar sulcus foveolate (Figs 5 and 6). Length of intercoxal space longer than fore coxae. Netrion (Fig 6) present as area of smooth surface sculpture, delimited dorsally by weak indication of netrion sulcus. Mesopleural pit (mspp Fig 6) deep, slightly transverse, with weak sulcus extending towards tegula. Acropleural sulcus (asu Fig 6) more or less continuous, the foveae almost completely merged; acetabular carina short, without post acetabular sulcus (i.e. no foveae present; ac Fig 4); postacetabular patch (papc Fig 6) clearly indicated, setose; episternal foveae absent; metapleural carina broadly foveolate (mc, Fig 6); metapleural sulcus (mtps Fig 6) indicated as a weak, shallow groove posterior to the metapleural pit (mtpp Fig 6); anteroventral surface of metapleuron with a series of fine grooves that extend towards the acutely pointed anteroventral extension (aem, Fig 6). Metascutellum (= dorsellum) with deep, regular reticulation in anterior half and weak longitudinal carinae in posterior half, interstices smooth (Fig 5).

Fig 5

Telenomus nizwaensis Polaszek male paratype.

Lateral mesosoma.

Fig 6

Telenomus nizwaensis Polaszek male paratype.

Dorsal mesosoma.

Metasoma (Fig 1) T1 with 1 pair of sublateral setae, 2 pairs of lateral setae. Basal costae on T1 reaching its posterior margin centrally; basal costae on T2 present as a row of foveae. Ventral metasoma (Fig 7) entirely smooth, a pair of large setae close to the posterior margin of S2; many smaller setae present on laterotergites centrally.

Fig 7

Telenomus nizwaensis Polaszek female paratype.

Ventral metasoma.

Genitalia (Fig 8). Central projection absent; digiti large relative to aedeagal lobe (about half its length); aedeagal lobe 0.40× length aedeagovolsellar shaft, truncate. Digiti with 3 digital teeth. Basal ring comprising 0.30× length of entire aedeagus.

Fig 8

Telenomus nizwaensis Polaszek male genitalia.

Variation. Length 0.71–0.79 mm. Extensive variation in colour with many specimens, including paratypes with the metasoma lighter than in the holotype. Morphologically extremely uniform.

Female: Morphologically similar to male with the main exception of the antenna. Clava present and 4-merous, papillary sensilla on A8(1); A9(2); A10(2) A11(1) (Figs 9–11). F1 slightly longer than wide, all other antennomeres wider than long, except A11 (terminal claval segment).

Fig 9

Telenomus nizwaensis Polaszek female paratype.

Terminal antennomeres under differential interference contrast. Papillary sensilla indicated by arrows.

Fig 11

Telenomus nizwaensis Polaszek female.

Detail of A7 showing lack of papillary sensilla.

Species-group placement. Telenomus californicus-group

Hosts. Known from the eggs of its natural host, D. livia (Lepidoptera: Lycaenidae). Observations of emergence from eggs of other hosts require confirmation.

Distribution. Oman.

Material examined: Holotype ♂: OMAN, Al Jabal Al Akhdar (part of the Hajar mountain range in Ad Dakhiliyah Governorate of Oman; capital city of Nizwa); June 2015 ex eggs of D. livia. R. Al Shidi and A. Al-Riyami col. DNA1309: A14; genitalia mounted separately on microscope slide (NHMUK 013377700; HYM 9.1024). Paratypes: 5♀ 20♂, same data as holotype (3♀ [2 sputter-coated for SEM] 10♂, NHMUK 013378214–03378227; 1♀ 1♂ ONHM, 1♀ 1♂ USNM).

Molecular analyses

Non-destructive extraction and sequencing of three T. nizawaensis specimens produced DNA fragments of 414 (28S) and 658 (CO1) nucleotides. The nucleotide sequence of each gene was identical in each of the three specimens. The sequence alignments produced with MAFFT were 475 (28S), 851 (CO1), and 1326 (28S+COI) characters in length (base pairs plus gaps). In all phylogenetic analyses, the three specimens of T. nizwaensis clustered together unequivocally with 100% bootstrap support (Fig 12). Sequence data associated with each specimen has been deposited in GenBank under accession numbers MT635051–MT635053 (CO1) and MT636558–MT636560 (28S).

Fig 12

RAxML analyses (CO1, 28S, CO1+28S).

28S D2 (nuclear ribosomal)

A blastn search of the T. nizwaensis 28S nucleotide sequence against the NCBI nt database produced two hits identical of max score (712) and percent identity (97.8) to undetermined Telenomus spp. (GenBank accessions JX683253 and JX683244). The closest described species with which T. nizwaensis matched was T. sechellensis (703 max score; 97.3% identity). Telenomus nizwaensis was recovered as sister to a grouping of T. goniopis, an unidentified Telenomus, T. busseolae, and T. sechellensis.

CO1 “barcode” (mitochondrial)

A blastn search of the T. nizwaensis CO1 nucleotide sequence against the NCBI nt database yielded best matches to Telenomus dignus KR270640 (859 max score), and Scelionidae sp. KM566105 and Scelioninae sp. HM883306 (92.1% identity). The phylogenetic analysis based on the CO1 sequences of T. nizwaensis and eight other species of Telenomus resulted in a sister group relationship between T. nizwaensis and T. consimilis + T.dolichocerus, albeit with low bootstrap support.

Discussion

Taxonomy

There are 628 described, extant species of Telenomus. Synonymies are therefore extremely likely to be detected in future, as happened recently (and spectacularly) in the closely related genus Trissolcus [10]. The possibility that T. nizwaensis was described previously under another name cannot be ruled out. However, the task of obtaining, or attempting to obtain, each Telenomus species holotype to exclude that possible species identity, would at least severely delay, and possibly prohibit, the execution of this work. The morphological and molecular data presented here permit the unequivocal identification of T. nizwaensis. Future studies, especially using newly developed technologies, may discover a senior synonym of T. nizwaensis.

The 4-merous clava in the female has not been observed previously in any Telenomus species of the T. californicus goup, all members of which have been assumed to have the clava 5-merous [9]. However, papillary sensilla can be extremely difficult to see, and the presence of a single sensillum on A7 may have been wrongly assumed in the past for some species.

Guided in part by the extensive collections of Old World Telenomus at NHMUK, together with groundwork provided by Johnson [9], it has been possible to eliminate all African and Asian species described by Nixon [24-30], as well as many others held in the NHMUK collection. There remain a great many species of Telenomus described by Kozlov and Kononova [31], some from Central Asia that could conceivably also be present in the Arabian Peninsula.

Biology and economic importance

Telenomus is a highly speciose genus of minute wasps in the family Scelionidae (included until recently in the family Platygastridae), all of which are endoparasitoids of eggs of several insect orders, including the Heteroptera, Diptera, Neuroptera and, especially, the Lepidoptera. Some species, such as Telenomus remus, the most common egg parasitoid of Spodoptera frugiperda in the Americas, are very well-known parasitoid and are mass-reared and deployed in augmentative biological control in many countries [32]. T. nizwaensis is not well known but it is a natural enemy of a pest of economic importance, and has clear potential as an indigenous agent of biological control in Oman, and may also prove effective elsewhere.

Field notes and samples collected from D. livia eggs suggest that its life-history is similar to other species in the subfamily Telenominae. For instance, females of Telenomus fariai start ovipositing hours after emergence [33] and similarly, newly emerged T. nizwaensis females attack fresh eggs of the host, D. livia (A. A-R. pers. obs.). T. nizwaensis is a solitary species: females lay a single egg per host egg, or at least only one adult emerges from each host, as reported for T. remus [34]. Telenomus adult usually emerge through a hole in the side of the host’s chorion [35] and similar emergence holes are observed for T. nizwaensis.

A constraint in studying T. nizwaensis is that successful laboratory rearing protocols have not yet been developed. Eggs of wild tiger moth Utetheisa sp., cabbage looper Trichoplusia ni, and cotton leafworm Spodoptera littoralis have been exposed to T. nizwaensis parasitoids by researchers in the Omani Directorate General of Agricultural and Livestock Researches of the Ministry of Agriculture and Fisheries (2014), but no T. nizwaensis emerged from any of these eggs. Deudorix livia migrates to Acacia sp. (locally called Talh) during mid-September, at the end of the pomegranate season, and Telenomus wasps that may have been T. nizwaensis have emerged from butterfly eggs infesting Acacia sp. pods (A. A-R. pers. obs.).

Current field data indicate that T. nizwaensis is the most dominant parasitoid of D. livia in the Al Jabal Al Akhdar region. Annual reports of the IPM program run by the Omani Directorate General of Agricultural and Livestock Researches of the Ministry of Agriculture and Fisheries show that the parasitism rate by T. nizwaensis over the past 15 years was 62%, compared with 8.2% by Trichogramma sp. Telenomus nizwaensis begins attacking D. livia later in the pomegranate fruiting season, with peak parasitism between mid-June and mid-July, when pomegranate trees start to flower at the end of April. Alternative control measures are likely to be needed if earlier damage by D. livia is to be avoided. Sex ratios of T. nizwaensis emerging from naturally collected D. livia eggs in Oman (temperature range 20–35°C, June-July 2020) have a female biased sex ratio (proportion of offspring that are male ≈ 0.37, A. Al-R. unpublished data). Female biased sex ratios are generally a positive attribute in term of a parasitoid’s ability to suppress pest populations because only the female adults attack the next generation of hosts, and fewer host resources were used for the production of males [36].

Conclusions

We have formally described a new species of scelionid wasp, Telenomus nizwaensis Polaszek. Its biology is similar to that of its congeners and, as a natural enemy of the pomegranate butterfly, D. livia, it is a beneficial component of pomegranate agro-ecosystems in Oman. Current studies are aimed at further evaluating and promoting its field performance as an agent of biological pest control.

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Taxa, GenBank accession numbers, species group placement, collection location, and references associated with the sequence data used in the phylogenetic analyses.

Species group/complex placement mainly follows Johnson (1984) and Taekul et al. (2013).

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7 Jan 2021

PONE-D-20-34715

Telenomus nizwaensis (Hymenoptera: Scelionidae), an important egg parasitoid of the pomegranate butterfly Deudorix livia Klug (Lepidoptera: Lycaenidae) in Oman

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Reviewer #1: In this manuscript, the authors describe a new species, Telenomus nizwaensis Polaszek sp. n., based on morphology and DNA sequence data. This egg parasitoid is an important biological agent against the pomegranate butterfly Deudorix (=Virachola) livia, which is a major pest of pomegranate in Oman. They also summarize the biology of the parasitoid species, which might enhance to the further use of this parasitoid in biological control programs. This study is quite straightforward and the manuscript is well written. There are a few points of consideration listed below that I think might improve the manuscript.

Major comments

1. In the method section, the authors say that eight individuals (4 females, 4 males) were subjected to DNA extraction (line 106), while in the result section they mentioned that 28S and COI were generated only from three specimens (line 208). So only these three specimens were successfully sequenced? What are the genders of these three specimens? It would be good to know that if these three specimens cover both sexes. Apparently, the studied specimens were reared from the eggs of the pomegranate butterfly from the same locality and it is mostly likely, the emerged parasitoids belong to the same species, but you never know, other species of Telenomus might present, too. If the sequenced specimens are both from female and male, then the association of both sexes can be sure.

2. The new species is placed in the Telenolmus californicus-group, I believe it would be useful to readers if the authors could provide more information on the diagnosis of this species group. Besides, since the new species has a 4-merous clava in the female, which is different from other species of the group, the placement in the group should be discussed more in more details. In other words, why do the authors believe this new species belong to the Telenolmus californicus-group?

3. The author stated that the CO1 sequence of the new species is best match to Telenomus dingus KR270640 (line 225), what is the identical percentage between these two species? And why the CO1 sequence of Telenomus dingus KR270640 was not included in the phylogenetic analysis?

4. Line 95-96, the authors state “Observations of emergence from eggs of other hosts require confirmation”, also line 277-288 “Telenomus wasps that may have been T. nizwaensis have emerged from butterfly eggs infesting Acacia sp. pods (A. A-R. pers. obs.)”. The confirmation of the host range of the new species is important to biological control programs, it would be better to include the DNA of specimens emerged from other hosts into the analysis. Of course, this is optional if there were no such specimens available.

Minor comments:

1. In figs 12-14, the first two trees indicated as RAxML, while the last is not, is it also RAxML?

2. Please provide the full descriptions for the abbreviations in figs 5-6, either in the main text or the legend.

3. It seems that there are duplicates of figs 9-10.

Reviewer #2: Telenomus is a large and difficult genus to study as the authors mentioned. The authors described a new parasitoid species with host information and DNA barcodes. The description of this new hymenopteran species will benefit the management of its host pest as a potential biological control agent, as well as facilitate the species delimitations/identifications of the genus Telenomus. This new species is placed in the californicus-group, however, the interpretation of this species group was not provided. Further, the sister grouping relationships were recovered in the phylogenetic analyses with limited data. However, the possibly close relationships of this new species with another species in the californicus-group were not discussed. The discussions about its close relationship with other species and the interpretation of the species group where it is placed could help readers to understand the systematic position of this species in this species group or even in this genus.

Please see the details as in the attached file.

**********

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: Telenomus nizwaensis 4.11.2020.docx

Click here for additional data file.

23 Feb 2021

PONE-D-20-34715

Telenomus nizwaensis (Hymenoptera: Scelionidae), an important egg parasitoid of the pomegranate butterfly Deudorix livia Klug (Lepidoptera: Lycaenidae) in Oman

PLOS ONE

RESPONSE TO REVIEWERS (our responses in blue text)

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2. We noticed you have some minor occurrence of overlapping text with the following previous publication, which needs to be addressed:

https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0223761

In your revision ensure you cite all your sources (including your own work), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

• Since the extraction + sequencing protocols, several imaging protocols and other aspects of the methods were exactly identical to those in previous publications, we see no reason to artificially modify them for the sake of making them read differently. However, we acknowledge that the previous +/- identical protocols should be cited. At the risk of over self-citation I have cited only https://doi.org/10.1371/journal.pone.0223761 (Goniozus omanensis) and not https://doi.org/10.1371/journal.pone.0230944 (Metaphycus macadamiae). Unfortunately this has necessitated re-numbering the references.

•

3. In your Methods section, please provide additional location information of the collection sites, including geographic coordinates for the data set if available.

• Done

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"We thank the Ministry of Agriculture, Fisheries and Water Resources Agricultural Wealth,

304 Fisheries and Water Resources in Oman for support, especially Naser Al Abri and Issa Al-Mandhari

305 for their help in field work while collection and for offering other essential logistics. A. A-R was

306 funded by the Sultanate of Oman, Ministry of Higher Education, Research and Innovation under

307 grant number PGE055580 D.P.S/321/2017. We thank the Anglo-Omani Society for assistance

8

with publication charges. We also thank the Department of 308 Agricultural Development in Al Jabal

309 Al Akhdar for their valuable help, in particular as the contact point between us and local farmers."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"A. A-R: PGE055580 D.P.S/321/2017

Sultanate of Oman, Ministry of Higher Education, Research and Innovation

https://www.mohe.gov.om

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

• I have removed the specific funding references for the Oman Ministry. Should I also do this for the publication costs?

5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

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We will update your Data Availability statement to reflect the information you provide in your cover letter.

• We have submitted the unaligned and aligned fasta files to Dryad. Those files can be accessed at the following link: https://doi.org/10.5061/dryad.sxksn0329.

6. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

• This should fine – I have been lead/corres[poning author on 2 PLoSOne papers since 2016

7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

• Done

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors describe a new species, Telenomus nizwaensis Polaszek sp. n., based on morphology and DNA sequence data. This egg parasitoid is an important biological agent against the pomegranate butterfly Deudorix (=Virachola) livia, which is a major pest of pomegranate in Oman. They also summarize the biology of the parasitoid species, which might enhance to the further use of this parasitoid in biological control programs. This study is quite straightforward and the manuscript is well written. There are a few points of consideration listed below that I think might improve the manuscript.

Major comments

1. In the method section, the authors say that eight individuals (4 females, 4 males) were subjected to DNA extraction (line 106), while in the result section they mentioned that 28S and COI were generated only from three specimens (line 208). So only these three specimens were successfully sequenced? What are the genders of these three specimens? It would be good to know that if these three specimens cover both sexes. Apparently, the studied specimens were reared from the eggs of the pomegranate butterfly from the same locality and it is mostly likely, the emerged parasitoids belong to the same species, but you never know, other species of Telenomus might present, too. If the sequenced specimens are both from female and male, then the association of both sexes can be sure.

This information has now been updated

2. The new species is placed in the Telenolmus californicus-group, I believe it would be useful to readers if the authors could provide more information on the diagnosis of this species group. Besides, since the new species has a 4-merous clava in the female, which is different from other species of the group, the placement in the group should be discussed more in more details. In other words, why do the authors believe this new species belong to the Telenolmus californicus-group?

• This point is very well-made. One of us (ZL) has thoroughly updated the discussion on the species-group placement after discussion with Prof. N. Johnson., the author of the “californicus complex”

3. The author stated that the CO1 sequence of the new species is best match to Telenomus dingus KR270640 (line 225), what is the identical percentage between these two species? And why the CO1 sequence of Telenomus dingus KR270640 was not included in the phylogenetic analysis?

• The analyses have been completed re-done. We included all available and appropriate sequnnces from GenBank as well as the sited sequence from Telenomus dignus, though that had to be extracted from a mitogenomic study. The results are much more detailed and rigorous, although conclusions from the first analysis are still completely supported. We have added a list of all sequences analysed, their depositories etc as supplementary files.

4. Line 95-96, the authors state “Observations of emergence from eggs of other hosts require confirmation”, also line 277-288 “Telenomus wasps that may have been T. nizwaensis have emerged from butterfly eggs infesting Acacia sp. pods (A. A-R. pers. obs.)”. The confirmation of the host range of the new species is important to biological control programs, it would be better to include the DNA of specimens emerged from other hosts into the analysis. Of course, this is optional if there were no such specimens available.

• Yes, that would me a “nice to have”, but considering how many 100s of undescribed Telenomus species there must be out there, simply not practically possible right now. At the very least, our thorough analysis shows that T. nizwaenesis does not cluster closely with any other known species.

Minor comments:

1. In figs 12-14, the first two trees indicated as RAxML, while the last is not, is it also RAxML?

2. Please provide the full descriptions for the abbreviations in figs 5-6, either in the main text or the legend.

3. It seems that there are duplicates of figs 9-10.

• All these points have been addressed

Reviewer #2: Telenomus is a large and difficult genus to study as the authors mentioned. The authors described a new parasitoid species with host information and DNA barcodes. The description of this new hymenopteran species will benefit the management of its host pest as a potential biological control agent, as well as facilitate the species delimitations/identifications of the genus Telenomus. This new species is placed in the californicus-group, however, the interpretation of this species group was not provided. Further, the sister grouping relationships were recovered in the phylogenetic analyses with limited data. However, the possibly close relationships of this new species with another species in the californicus-group were not discussed. The discussions about its close relationship with other species and the interpretation of the species group where it is placed could help readers to understand the systematic position of this species in this species group or even in this genus.

• All points well-made and addressed thoroughly in our revision – see also the points able

Please see the details as in the attached file.

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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Submitted filename: Response to reviewers.docx

Click here for additional data file.

7 Apr 2021

Telenomus nizwaensis (Hymenoptera: Scelionidae), an important egg parasitoid of the pomegranate butterfly Deudorix livia Klug (Lepidoptera: Lycaenidae) in Oman

PONE-D-20-34715R1

Dear Dr. Polaszek,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Feng ZHANG, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

12 Apr 2021

PONE-D-20-34715R1

Telenomus nizwaensis (Hymenoptera: Scelionidae), an important egg parasitoid of the pomegranate butterfly Deudorix livia Klug (Lepidoptera: Lycaenidae) in Oman

Dear Dr. Polaszek:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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