Biology and management of Coleophora deauratella (Lepidoptera: Coleophoridae) in red clover seed-growing regions in North America and New Zealand

Abstract

Red clover (Trifolium pratense L.), a perennial forage legume belonging to the Fabaceae family, is grown for seed in many temperate regions of the world. Coleophora deauratella Leinig and Zeller (Lepidoptera: Coleophoridae) is a significant insect pest occurring globally in several primary red clover seed-producing regions. Coleophora deauratella inflicts crop damage by larval feeding on developing seed within individual florets, thus reducing seed yield. The first detection of C. deauratella and seed yield losses up to 90% were reported in the Peace River region of Alberta, in western Canada, in 2006, signifying its damage and potential threat to other red clover seed-producing areas of the world as an invasive insect pest species. As a result, crop stand age was reduced to 1 yr to mitigate seed yield loss caused by this pest in second-year fields in Alberta. Coleophora deauratella was first discovered in western Oregon in 2011, but the resulting economic damage remains unknown after more than a decade of its discovery. The first confirmed case of C. deauratella and tremendous seed yield devastation in red clover seed crops in the mid-Cantebury region of New Zealand occurred in 2016. Continued monitoring efforts in Oregon and New Zealand revealed that pest populations started receding after 2018, and the presence of unknown biocontrol agents, climatic, or genetic factors was speculated for its lower establishment rate. In this article, we discuss C. deauratella biology, ecology, and pest status in North America and New Zealand, along with the key research highlights to control C. deauratella.

Introduction of Coleophora deauratella in North America and New Zealand

Red clover (Trifolium pratense L., Fabaceae) is an important short-lived perennial legume crop grown for seed in the United States, Canada, Europe, and New Zealand (Fairey 1981, Taylor and Quesenberry 1996). Inadequate pollination and increasing pest pressure are the primary reasons for declining seed yields worldwide (Vleugels et al. 2019). The Willamette Valley of Oregon is one of the major producers of red clover seed for the global market, with over 8.1 thousand ha and generating approximately US$11.5 million in total production value (Anderson et al. 2020, Anderson 2022). Insect pests and pathogens are the primary factors that inflict significant reductions in seed yield in older stands, resulting in seed producers limiting the stand age for red clover seed fields in Oregon between 2 and 3 yr (Oliva et al. 1994, Steiner and Alderman 1999, Anderson, unpublished data).

Coleophora deauratella Leinig, and Zeller (Lepidoptera: Coleophoridae) is an invasive insect pest species that threatened the profitability of red clover seed production in North America and New Zealand in the last few decades (Evenden et al. 2010, Anderson et al. 2014, Chynoweth et al. 2018, Walenta et al. 2019, 2020, Kaur et al. 2021). Originating from Europe, eastern Serbia, and the Middle East (Landry and Wright 1993, Ellis and Bjørnson 1996, Haye et al. 2015, Mori et al. 2016), C. deauratella is not considered an economic insect pest in the areas of its origin. Coleophora deauratella is among the multiple species of the case-bearing moths in the family Coleophoridae (Heinrich 1923, Pearson 1975, Emmet et al. 1996) that were introduced to North America and New Zealand, causing tremendous seed yield loss immediately after their introduction (Pearson 1989, Chynoweth et al. 2018). The members of this genus derive their common name due to the behavior of mature larval instars that construct and inhabit a portable silk case made of plant material such as entire florets, bracts, leaf edges, seeds, or even sand.

In addition to C. deauratella, 2 other invasive species of European origin in the genus Coleophora introduced to North America are C. mayrella (Hübner) and C. trifolii (Curtis), which are host specific to other members of the plant family Fabaceae, including white clover (T. repens L.) and sweet clover (Melilotus sp.), respectively (Landry and Wright 1993, Evenden et al. 2010). The earliest record of C. deauratella in North America is in Ithaca, NY, in the 1960s. Still, it was not recognized until 1990 due to a case of incorrect identification and confusion between specimens of closely related species (C. mayrella and C. trifolii) (Landry 1991, Ellis and Bjørnson 1996). Initially, the distribution of C. deauratella in North America was thought to be restricted to the northeastern United States and southeastern Canada as of 1993 (Landry and Wright 1993). The first report of this pest in western North America came from Alberta in 2006 (Evenden et al. 2010) and has since become a significant pest of clover grown for seed in this region.

In New Zealand, 2 species of clover-feeding moths of the Coleophora genus, Coleophora mayrella and C. alcyonipennella Kollar, have seriously hindered red and white clover seed production for more than a century (Pearson 1975, Chynoweth et al. 2018; Faulkner, unpublished data). Areawide biocontrol efforts were initiated in the 1960s to introduce and establish 2 biological control agents that significantly reduced the negative effect of these pests (Pearson 1989). This program was highly successful, resulting in the total suppression of clover casebearer seed pests in New Zealand until the arrival of C. deauratella in 2016. This species quickly spread throughout the country’s clover-growing regions, leading to yield reductions (Faulkner, unpublished data). Over this period, severe seed yield losses were recorded, especially in third- and fourth-year red clover stands (Chynoweth et al. 2018).

In this article, we discuss the biology and ecology of C. deauratella in both North America and New Zealand, along with its current pest status and some management options.

Economic Impact of Coleophora deauratella in North America and New Zealand

In 2006, 90% yield loss was documented in the second- and third-year fields due to C. deauratella in the Peace River region of Alberta in western Canada, limiting stand longevity in perennial red clover grown for seed to an annual cycle (Evenden et al. 2010). Utilizing a sex pheromone (10:1 ratio of (Z)-7-dodecenyl acetate [Z7-12:OAc] to (Z)-5-dodecenyl acetate [Z5-12:OAc]) (Evenden et al. 2010, Mori 2014, Mori et al. 2014) in an attractant-based trap network (Fig. 1) is the primary mechanism used to monitor the distribution and range expansion of the pest across the production region and investigate seasonal phenology of adult populations in red clover production areas worldwide.

The detection of C. deauratella in Oregon’s Willamette Valley in 2011 and the successful establishment of this pest were confirmed by subsequent monitoring efforts (Anderson et al. 2014, Walenta et al. 2019, 2020, Kaur et al. 2021). Shortly after, C. deauratella was detected in red clover fields of Grande Ronde Valley in northeastern Oregon and in the Treasure Valley on the eastern edge of Oregon in 2018 and 2019, respectively (Walenta et al. 2019, 2020). Although high adult densities were present in pheromone-baited traps, corresponding seed yield loss due to larval feeding has not been confirmed in Oregon (Kaur et al. 2021). Currently, there are only anecdotal reports suspecting C. deauratella infestations causing significant seed yield loss in Oregon red clover crops (Anderson, unpublished data). However, reduced stand longevity and significant seed yield loss reports in second-year stands may be due, in part, to undetected C. deauratella feeding damage. The potential economic impact of this invasive pest species on Oregon’s seed industry in the future underscores the need to improve understanding of C. deauratella biology, geographic distribution, and potential host range, as well as determining effective management tactics to reduce the severity of outbreaks on crop productivity.

New Zealand’s South Island represents another recently invaded red clover seed production region (~1,000 ha annually), where C. deauratella was first detected in 2016. Significant seed yield reductions similar to losses reported in the Peace River region of Alberta, Canada, occurred in the mid-Canterbury area during the 2016–2017 growing season (Chynoweth et al. 2018). Over this period, severe seed yield losses (80%–99.5%) were recorded, especially in third- and fourth-year red clover stands (Chynoweth et al. 2018). Since the discovery of C. deauratella in New Zealand, considerable research has been initiated to determine integrated management strategies, including potential biocontrol agents, chemical control timing and product selection, and reducing nontarget impact on beneficial arthropod communities (Chynoweth et al. 2018, Rolston et al. 2019).

Description and Life Cycle

Typically, C. deauratella moths are active during dawn, and flights occur during summer (Fig. 2a). Adult activity occurs from June through mid-August in red clover seed-growing regions of Oregon and Alberta (Anderson et al. 2014, Mori et al. 2014, Dorman et al. 2023) and from mid-November to mid-January in New Zealand (Chynoweth et al. 2018, Rolston et al. 2019), respectively (Fig. 2b). After mating, female moths lay white eggs directly on the calyx of florets of newly set red clover inflorescences. Eggs are laid individually on the floret, and subsequent larval populations enter the floret after eclosion by chewing holes through the corolla and feeding on the developing seed (ovule) (Figs. 2a and 3a). There are 4 instars. The first through second instars are concealed within original florets of red clover inflorescence feeding on the seed (Fig. 3b). The third instar consumes any remaining seed within the original floret before boring out of the floret, creating an exit hole, and penetrating into the ovules of adjacent florets (Walenta et al. 2019, Fig. 3f). These first 3 instars are very rarely observed in the field, as they remain entirely concealed within the inflorescence (Walenta et al. 2019). The fourth instar is detected more easily since they construct a protective case of floret parts over their body while actively feeding on developing seeds. The features of the case are a round feeding hole from which the front of the larva emerges to feed and move around (Fig. 3c), and the distal end that is made of 3 trilobite flaps that can be opened for removing frass and other debris (Pearson 1975). Larvae are generally difficult to identify using morphological characters (Landry and Wright 1993, Bauer et al. 2012), and molecular methods are commonly used for species identification and distinction (Landry and Wright 1993, Bauer et al. 2012). The mature larvae with the protective cases (Fig. 3c) then crawl to the ground as the inflorescences senesce close to the crop harvest period, seal themselves (Fig. 3d), and overwinter on the soil surface or within crop residues. Warming spring temperatures cue the termination of larval diapause, triggering pupation, followed by adult emergence in early summer (Ellis and Bjørnson 1996).

The adult moths of C. deauratella are tiny with shiny metallic wings spanning 11–13 mm in length (Landry and Wright 1993, Cottrell 2018) (Fig. 3e). The forewings have a metallic color with fringed hairs stretching to the posterior tip of the wing, and the hindwings are dark-colored with fringed hairs longer than the forewings; fringed hairs are also observed near the eyes. The base of the antennae is thickened with projecting scales and is longer in female moths than in male moths (Landry and Wright 1993, Cottrell 2018).

Dispersal and Risk Factors

There are several lines of evidence that suggest human-assisted transport likely contributed to the intracontinental movement of this species. The short adult longevity, univoltine life cycle, and small wingspan (Landry 1991, Mori and Evenden 2015) likely limit C. deauratella’s ability to move long distances. Mori et al. (2016) studied the genetics of C. deauratella populations in North America. This research revealed a lack of genetic diversity that suggests that populations were founded by a small number of individuals (founder effect). Research by Mori et al. (2016) investigated the possible origin of C. deauratella populations in North America to speculate on potential biological control agents associated with its source habitat. This work suggested the probable origin of North American C. deauratella populations was Switzerland based on similarities of the mitochondrial COI region with Swiss populations, but acknowledged that more sampling in the native region is necessary to confirm the true origin (Mori et al. 2016). Further loss of genetic differentiation and cluster analyses showed a lack of isolation by distance in North American populations. These findings suggest the introduction of C. deauratella in Ontario, Canada, or adjacent US states and subsequent dispersal across the continent occurred by accidental movements of contaminated agricultural products or trade.

Interestingly, in the recent past, C. deauratella pressure began receding across Oregon (2020 onwards) (Fig. 4). Similarly, C. deauratella abundance data collected from the red clover seed-producing areas of New Zealand (Fig. 4) during the 2019–2020 and the 2020–2021 growing seasons indicated the pest pressure had diminished significantly when compared with the monitoring data collected during 2017–2018 (Chynoweth et al. 2018). This unexpected population decline points to the potential presence of an unknown biological control agent, climatic, or other genetic factors preventing the pest from reaching its damaging potential in the areas of its new introductions.

Crop defoliation for synchronous flowering or cutting of the red clover silage is a unique agronomic practice in Willamette Valley of Oregon and might be hampering larval feeding early on and preventing damage to seeds during the bloom and seed fill period (Anderson et al. 2014, Walenta et al. 2019, 2020). Thus, an unintentional mechanical control of C. deautratella occurs in Oregon red clover fields, followed by sanitation or clean-up when fields are cut (flailed or swathed) for silage (Anderson et al. 2020). However, when plants are cut to delay flowering rather than for silage, crop residues, including the clover inflorescence infested with casebearer larvae, are left on the ground. These larvae can overwinter in first-year clover stands and complete their life cycle the following year, contributing to greater damage in second-year stands; greater populations of C. deautratella have been documented in second-year stands in all affected regions (Canada, Oregon, New Zealand) (Mori et al. 2014, Chynoweth et al. 2018, Walenta et al. 2019).

Host Range

Coleophora deauratella is known to feed on multiple host plants within the plant family Fabaceae, mainly Trifolium species, including T. arvense (stone clover), T. hybridum (alsike clover), T. medium (zig-zag clover), T. pratense (red clover), and T. repens (white clover) (Hammer 1937, Markkula and Myllymäki 1960). Trifolium pratense (red clover) is the preferred host plant (Hammer 1937, Markkula and Myllymäki 1960). A DNA-based method known as gut content analyses (Cooper et al. 2019) that amplifies regions of plant-derived internal transcribed spacer and the chloroplast trnL genes was used to infer their host range or dietary history of the early season field-collected C. deauratella adults from the commercial red clover fields in Willamette Valley of Oregon (Kaur et al. 2021, Dorman et al. 2023). Most of the sequence data obtained in this study corresponded to the plant family Fabaceae (85.7% Trifolium species, 28.6% Vicia species) (Kaur et al. 2021, Dorman et al. 2023). Pheromone-based traps deployed in commercial fields of 2 Vicia species later confirmed the presence of C. deauratella adults in hairy vetch, Vicia villosa Roth, and common vetch, Vicia sativa L. crops in the Willamette Valley in 2021 (Kaur, unpublished data).

Nature of Injury

Symptomology of C. deauratella feeding damage in red clover includes larval entry holes on the florets of the inflorescence (Fig. 3f). Third instars move throughout the inflorescence when feeding, and one larva can consume 2–3 developing seeds per day, leading to 80%–99% seed loss in red clover seed-growing regions of Canada (Landry 1991, Ellis and Bjørnson 1996, Evenden et al. 2010). Similar patterns of seed yield loss were reported in New Zealand in 2018, especially in third- and fourth-year stands (Chynoweth et al. 2018). The seed yield loss potential by C. deauratella in Oregon is still unknown. Large adult captures (>1,000 moths per pheromone-baited trap) were captured when traps were deployed in 8 commercial red clover seed production field sites in eastern Oregon during the 10-wk monitoring period in the years 2018 and 2019, but corresponding seed damage was not reported (Walenta et al. 2019, 2020) similar to the years 2013 and 2014 Willamette valley red clover seed field monitoring surveys (Fig. 4). Destructive sampling of inflorescences revealed an extremely low level of larval infestation, indicating the potential disruption in the pest’s life cycle by mechanical or biological control factors.

Since the introduction of C. deauratella in Oregon and New Zealand, collaborative research projects have been developed in North America and New Zealand with the main emphasis on understanding the pest’s current distribution, investigating the host plant range or dietary history of newly emerged moths using gut content analyses (Kaur et al. 2021), and testing landscape-level effects for geospatial and phenological modeling to develop a risk prediction framework to mitigate outbreak severity (Dorman et al. 2023). Ongoing collaborative research efforts in recent years enhanced our understanding of the biology and ecology of C. deauratella to fill in some of the critical knowledge gaps for developing sustainable management strategies against this pest.

Management Considerations

Action threshold levels do not currently exist for C. deauratella management in red clover seed crops. Potential management tools include relying on pheromone-based monitoring methods for early warning of adult flights and timing of C. deauratella outbreaks, understanding the geographic distribution and spread, and optimizing the timing of control measures, including chemical control and mating disruption (Evenden et al. 2010, Mori et al. 2014, Dorman et al. 2023). Adult flight activity information, developmental rate of immature life stages, and timing of associated feeding damage can help provide a comprehensive risk assessment for this pest throughout the growing season. In a 3-yr study on pheromone-baited trapping in red clover seed production in fields in Alberta, Canada, from 2010 to 2012, adult male densities were positively related to larval abundance and seed damage at moderate and high infestation levels (Mori et al. 2014). Another use of pheromone-based monitoring efforts will be to develop and extend existing seasonal phenology models for adult populations to the crop-damaging larval life stage based on cumulative growing degree days (GDDs) as a decision support system to optimize the timing of foliar insecticide applications (Ellis and Bjørnson 1996, Evenden et al. 2010).

Phenology Models

Accurate seasonal phenology models are necessary to predict the time of appearance of specific life stages to improve the timing of control tactics. A baseline temperature of 12.3 °C, the lower developmental threshold (LDT), and a start date of 1 January were used to calculate the cumulative GDDs and predict the seasonal spring flight of C. deauratella in Oregon (Dorman et al. 2023). Median moth flight activity for the Peace River region of Canada was reported to occur at 258 GDDs using an LDT of 11.7 °C (Mori et al. 2014), which aligned closely with the predicted median moth flight activity period in Oregon’s Willamette Valley at 256 GDDs with the start date of 1 January, albeit a slightly higher LDT was used based on extensive C. deauratella trap data collected in Oregon (Dorman et al. 2023). For New Zealand, the median flights occurred at 222 GDDs with a start date of 1 November. Both phenology models from Oregon and New Zealand performed with high accuracy levels to predict 25%, 50%, and 75% flight activity levels of C. deauratella adults.

Mating Disruption

Protracted adult flights for up to 6–8 wk during the bloom and seed fill period may require multiple insecticide applications to control C. deauratella, which poses a risk to both pollinators and other beneficial insects that are vital for maintaining seed yields in red clover seed crops (Mori and Evenden 2013). The univoltine and protandry nature of C. deauratella is advantageous for using pheromone-mediated mating disruption techniques for effective pest management (Mori and Evenden 2015). Mating disruption methods are species specific and do not interfere with nontarget organisms, especially pollinators like honey bees, Apis mellifera L., and native bumblebees, Bombus spp. L., providing essential pollination services for Oregon’s clover seed crops. Different pheromone release methods disrupted C. deauratella communication and mating in field trials conducted in commercial red clover seed crops in Alberta, Canada (Mori and Evenden 2015). Potential challenges with adopting mating disruption as a primary management solution for C. deauratella may include the immigration of gravid females into red clover stands combined with high population densities (Mori and Evenden 2014a, 2014b, 2015). Work by Kaur et al. (2021) observed up to 38% reduction in adult moth captures in pheromone traps and a 73% decline in subsequent damage in larval feeding in Oregon’s Willamette Valley, demonstrating the effectiveness of mating disruption for C. deauratella management. Continued testing will be needed to calibrate the timing, release rate, and release method of mating disruption techniques in Oregon and New Zealand.

Biological Control

Certain generalist predators (e.g., Orius species) and parasitoids appear to provide some degree of biological control. Historically, only 2 species of clover casebearer moths, the whitetipped clover casebearer (Coleophora alcyonipennella Kollar) and banded clover casebearer (C. mayrella Hubner), were pest species of concern in white clover seed crops in the 1950s–1970s in New Zealand (Stuart 1958, Trought 1979, Pearson 1980) and were largely controlled by classical biological control (Pearson 1989). Many species of parasitoids were introduced from Europe into New Zealand in the 1950s and 1960s, and 2 parasitoids, Neochrysocharis formosa (Hymenoptera: Eulophidae) and Bracon variegator (Hymenoptera: Braconidae), have successfully established (Haye et al. 2015). Three generalist parasitoid species, B. variegator, Pteramalus puparum L. (Hymenoptera: Pteromalidae), and Eupelmus (Macroneura) messene Walker (Hymenoptera: Eupelmidae) (Fig. 5), were found to be associated with fourth instars of the C. deauratella collected from 2 red clover seed fields in New Zealand (Faulkner, unpublished data).

In Ontario, Canada, N. formosa was released as a potential control agent for C. deauratella, but the parasitoids were not recovered from the pest; however, another parasitoid, Bracon pygmaeus (Hymenoptera: Braconidae), was recovered in Canada from parasitized C. deauratella (Ellis and Bjørnson 1996).

Generalist parasitoid wasps in Ptermomalidae and Icneumonidae families were predominant in the sweep net samples during field surveys of commercial red clover seed fields in Willamette Valley, Oregon, in 2020 and 2021, indicating their biocontrol potential (Kaur et al. 2021).

Chemical Control

Concealed and internal feeding habits of larvae present a unique challenge, generally inaccessible to nonsystemic or contact chemicals and a range of biocontrol agents (especially generalist predators). Only a few insecticide chemistries registered in clover seed crops grown in Oregon (Anderson 2023) are effective at targeting actively feeding larvae burrowed within the florets. Future implications of a recent ban on chlorpyrifos in Oregon on C. deautratella suppression are yet to be determined. Insecticide efficacy field trials in Alberta, Canada, reported no yield improvements using foliar-applied broad-spectrum insecticides to control C. deautratella (Otani, unpublished data). Insecticide application at the bud or the early flowering (adult flight period) stages was somewhat effective against adults (Otani, unpublished data). Chynoweth et al. (2018) reported high efficacy of synthetic pyrethroids (Tau-fluvalinate and lambda-cyhalothrin) and an organophosphate (chlorpyrifos) against adult moths in laboratory bioassays in New Zealand and subsequent studies to evaluate nontarget effects of these chemistries on pollinators, parasitoids, and pathogens are in progress.

Conclusion

Coleophora deautratella has not yet attained critical pest status in Oregon and New Zealand, and populations are possibly in decline. Vigilant monitoring and a sound understanding of pest biology and ecology in the introduced environment are essential to formulate preventative strategies to mitigate seed yield loss caused by this pest in the red clover seed-producing regions of the world (Kaur et al. 2021, Dorman et al. 2023). Prevailing mechanical, biological control, or genetic factors in Oregon and New Zealand red clover production systems may have suppressed C. deautratella populations below economically damaging levels in recent years. Furthermore, C. deauratella appears to have succumbed to some ecological or genetic pressure within the last 5 years, which has greatly lessened its impact as a pest in both Oregon and New Zealand. It is also possible that more typical genetic processes may be in play, such as inbreeding depression, which can quickly establish following the colonization limits of a pest being reached (Mori et al. 2016). Considering the substantial economic impact of C. deauratella, regular pest monitoring is still necessary for red clover seed production systems in Oregon and New Zealand. In preparation for potential C. deauratella outbreaks in future years, and to fill current knowledge gaps, continued research efforts can be directed to refine phenology models for predicting larval emergence and feeding damage timing as the current phenology model is based on the adult abundance. More research is also needed to calibrate the mating disruption technique and its utilization as a practical option for C. deauratella management in Oregon. Future trials can be designed to determine optimum application timing using both conventional chemical control options and mating disruption techniques against C. deauratella, integrating phenology models as early warning systems. Steps for the enhancement of biocontrol will provide additional pest management solutions in both Oregon and New Zealand in the future.

Acknowledgments

The authors acknowledge the funding from the Oregon Clover Commission and the Agricultural Research Foundation for supporting research efforts to understand the biology and ecology of C. deauratella in Oregon red clover seed production systems. We thank various grower cooperators, crop consultants who participated in this study, and technical staff for their assistance.

Author Contributions

Navneet Kaur (Conceptualization [Lead], Formal analysis [Lead], Funding acquisition [Lead], Investigation [Lead], Methodology [Lead], Project administration [Lead], Writing—original draft [Lead], Writing—review & editing [Lead]), Nicole Anderson (Conceptualization [Lead], Funding acquisition [Lead], Investigation [Lead], Methodology [Lead], Project administration [Lead], Writing—original draft [Lead], Writing—review & editing [Lead]), Seth Dorman (Conceptualization [Equal], Formal analysis [Equal], Funding acquisition [Equal], Investigation [Equal], Methodology [Equal], Writing—review & editing [Equal]), Darrin Walenta (Conceptualization [Equal], Data curation [Equal], Funding acquisition [Equal], Investigation [Equal], Methodology [Equal], Project administration [Equal], Writing—original draft [Equal], Writing—review & editing [Equal]), Brian Donovan (Data curation [Equal], Methodology [Equal]), Christy Tanner (Investigation [Equal], Methodology [Equal], Writing—review & editing [Equal]), Boyd Mori (Conceptualization [Equal], Data curation [Equal], Formal analysis [Equal], Investigation [Equal], Methodology [Equal], Writing—original draft [Equal], Writing—review & editing [Equal]), Jennifer Otani (Conceptualization [Equal], Data curation [Equal], Investigation [Equal], Methodology [Equal], Project administration [Equal], Writing—review & editing [Equal]), Richard Sim (Writing—review & editing [Equal]), Phil Rolston (Conceptualization [Equal], Funding acquisition [Equal], Investigation [Equal], Methodology [Equal], Project administration [Equal], Writing—original draft [Equal], Writing—review & editing [Equal]), and Joel Faulkner (Conceptualization [Equal], Data curation [Equal], Formal analysis [Equal], Investigation [Equal], Methodology [Equal], Writing—original draft [Equal])

References