Nematodes (roundworms), a group of non-segmented worm-like invertebrates, are some of the most abundant animals on earth in terms of individuals (Lorenzen, 1994; Poinar, 2011). They are distributed worldwide in almost all habitats and play key roles in ecosystems by linking soil food webs, influencing plant growth and facilitating nutrient cycling (Yeates et al., 2009; van den Hoogen et al., 2019; Zhang et al., 2020). The earliest known definite nematode fossil occurs in the Lower Devonian Rhynie Chert inside the cortex cells of an early land plant and has been considered to be a plant parasite (Poinar et al., 2008), but unconfirmed nematode-like fossils and trace fossils may date back to the Precambrian (Poinar, 1979; Parry et al., 2017; De Baets et al., 2021a). Despite their abundance in many extant ecosystems, nematodes are exceedingly rare in the fossil record, since most of them are small, with soft bodies and concealed habits (Poinar, 2011).

The Mermithidae represent a family of nematodes that are obligate invertebrate parasites which occur in insects, millipedes, crustaceans, spiders, molluscs, and earthworms (Nickle, 1972; Poinar, 1979). They can affect the morphology, physiology, and even the behavior of their hosts (Petersen, 1985). The life cycle of mermithids comprises five stages (Poinar and Otieno, 1974; Poinar, 1983 ; Poinar, 2001b). Eggs are deposited in the environment, and the developing embryos moult once and emerge from the eggs as second-stage juveniles. These juveniles are the infective stage that enter the hemocoel of potential hosts. After a relatively rapid growth phase (third stage), mermithids exit hosts as postparasitic juveniles (fourth stage). They then enter a quiescent phase and moult twice to become adults (Poinar, 2015a). They tend to exit their hosts even before maturation, though, if their hosts are stressed (Poinar, 2015b). Hosts usually die when the mermithids exit, which is why mermithids have been widely studied as possible biological control agents, especially against aquatic stages of medically important insects like mosquito larvae (Petersen, 1985). Although mermithids kill their hosts like parasitoids, they are commonly considered as parasites like other nematodes (De Baets et al., 2021a; De Baets et al., 2021b).

Palaeontological records of parasitism provide critical clues about the origination and diversification of important nematode groups, and elucidate the synecology and coevolution of ancient parasites and their hosts. Due to their relatively large size and invertebrate-parasitic habits, mermithid nematodes are most likely of all nematodes to occur as recognizable fossils, especially in amber as they exited their invertebrate hosts that became entrapped in resin (Poinar, 2011). Their fossil record dates back to the Early Cretaceous with Cretacimermis libani from a chironomid midge (Diptera: Chironomidae) in Lebanese amber (~135 Ma; million years ago) (Poinar et al., 1994). However, there is a dearth of examples of other Cretaceous mermithids prior to the present study, with only Cretacimermis chironomae, also from chironomid hosts, Cretacimermis protus from biting midges (Diptera: Ceratopogonidae) and Cretacimermis aphidophilus from an aphid (Hemiptera: Burmitaphididae) in mid-Cretaceous Kachin amber (Poinar and Buckley, 2006; Poinar and Sarto i Monteys, 2008; Poinar, 2011; Poinar, 2017).

Here, we report 16 additional mermithid nematodes associated with their insect hosts in mid-Cretaceous Kachin amber (approximately 99 million years old). These examples triple the diversity of Cretaceous mermithids (from 4 to 13 species) and reveal previously unknown host–parasite relationships. Our study also shows that mermithids were widely distributed in a number of diverse insect lineages by the mid-Cretaceous, but they preferred to parasitize non-holometabolous rather than holometabolous insects, thus providing novel insights into the early evolution of nematode parasitism of insects.

The sixteen new mermithids associated with their insect hosts described above include 10 insect–mermithid associations. The hosts of nine species were previously unrecorded, which triples the diversity of Cretaceous Mermithidae (from 4 to 13 species). In today’s ecosystems, mermithids have been reported from a variety of arthropods, in a range of environments, and often infecting large percentages of host populations and causing mass mortality (Poinar, 1975; Poinar, 1979; Petersen, 1985). Despite their abundance in extant terrestrial ecosystems, mermithids are rare in the fossil record as they are not readily preserved as fossils. Twenty-two fossil mermithid species have been described from the Cenozoic with their hosts (Supplementary file 1: Table S1), mainly from Eocene Baltic amber (11 species) and Miocene Dominican amber (9 species), but only four pre-Cenozoic species associated with only two insect orders have previously been recorded (Poinar and Buckley, 2006; Poinar and Sarto i Monteys, 2008; Poinar, 2011; Poinar, 2017). However, according to our new records, nine insect orders are now known to have been infested by mermithid nematodes in Kachin amber and this number is even higher than that of Baltic amber (~45 Ma) and Dominican amber (~18 Ma) (six and three insect orders, respectively), despite a much longer time spent searching for nematodes in the latter two amber deposits (Poinar, 2011). Together with previously described mermithids in Kachin amber (Poinar, 2001b; Grimaldi et al., 2002; Poinar and Buckley, 2006; Poinar, 2011; Poinar, 2017), our results suggest that mermithid parasitism of insects was actually widespread during the mid-Cretaceous (Figure 8). Mermithids species are usually characterized by strong host specificity, they are specific to a single species or to one or two families of insects, and are almost always lethal to their hosts (Stoffolano, 1973; Petersen, 1985), thus our study indicates that the widespread mermithid parasitism probably already played an important role in regulating the population of insects in Cretaceous terrestrial ecosystems.

Figure 8

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Our study provides new information on fossil host–parasite associations, including three previously unknown host–mermithid associations and first fossil records of four host associations. One is Cretacimermis incredibilis sp. nov., which has completely exited from a bristletail (Archaeognatha). Its tail end is still adjacent to an exit wound on the host (Figure 2D), indicating a true parasitic association. There are no previous extant or extinct records of nematodes attacking bristletails (Poinar, 1975; Poinar, 2011). A second new mermithid–host association is barklice (Psocodea) with three different specimens parasitized by mermithids. No barklice are parasitized by mermithids today (Poinar, 1975), but our specimens imply that such relationship might have been quite common in the mid-Cretaceous. Two members of the extinct planthopper family Perforissidae were also parasitized by mermithids, thus providing the oldest record of mermithid parasitism of planthoppers. The mermithid Heydenius brownii parasitized achiliid planthoppers in Baltic amber (Poinar, 2001a) and this association also occurs in extant planthoppers (Choo et al., 1989; Helden, 2008). Furthermore, our findings are the first fossil records of mermithids parasitizing dragonflies (Odonata), earwigs (Dermaptera), crickets (Orthoptera) and cockroaches (Blattodea), four host associations predicted from extant records (Poinar, 1975).

Nematode body fossils are scarce and mainly known from amber (De Baets et al., 2021b), sometimes together with their hosts (Poinar, 2011). To explore the evolution of nematode–host relationship, we compiled nematode–host records in the three best-studied amber biotas (mid-Cretaceous Kachin amber, Eocene Baltic amber and Miocene Dominican amber; Supplementary file 1: Table S1). Our results indicate that not only the mermithids, but also the nematodes as a whole, experienced a certain degree of host transition between the Cretaceous and Cenozoic (Figure 9). We cannot fully exclude the possibility of collection bias, but its influence is probably low because Kachin amber has been extensively studied in the last two decades and its biota has already become the most diverse known amber biota; moreover, holometabolous insects are much more diverse in the collections than non-holometabolous ones (1296 vs 465 species: Ross, 2023). It is therefore unlikely that holometabolous insects are underrepresented among the known hosts of mermithids. Among the insect hosts of mermithids preserved in Kachin amber, only one of the nine orders (Diptera) is holometabolous (i.e. insects with ‘complete’ metamorphosis), whilst it is four out of six (Hymenoptera, Trichoptera, Lepidoptera and Diptera) in Baltic amber and all three insect host orders (Hymenoptera, Coleoptera and Diptera) are holometabolous in Dominican amber. The situation is similar when referring to the amount of nematode parasitism (Table 1). In Kachin amber, only about 40% of the hosts (in total, not only insects) are holometabolous, while this percentage increases to 80% in Baltic and Dominican amber and this result is acceptable when uncertainty is considered (Figure 9B). Diptera are the most common hosts of nematodes from all three amber biotas; also, the oldest fossil animal that was found to host a mermithid is a dipteran from Early Cretaceous Lebanese amber (Poinar et al., 1994). This is probably because most dipteran larvae develop in moist or aquatic environments that are particularly suitable habitats for nematodes (Poinar, 2011). It is evident that Holometabola are the most important hosts of extant mermithids as well as all invertebrate-parasitizing nematodes (Poinar, 1975) and this hexapod subgroup dominated the insect fauna during the Cretaceous (Labandeira and Sepkoski, 1993; Labandeira, 2005; Sohn et al., 2015; Peters et al., 2017; Zhang et al., 2018; Thomas et al., 2020; Wang et al., 2022). Our study suggests that, except for Diptera, nematodes had not completely exploited Holometabola as hosts in the mid-Cretaceous. This suggests that non-holometabolous insects (i.e. insects without ‘complete’ metamorphosis) were more available as hosts in the mid-Cretaceous and the widespread association between nematodes and Holometabola might have formed later.

Figure 9

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Finally, discovering these nematodes in mid-Cretaceous Kachin amber brings new opportunities to study the evolution of parasitism through the medium of amber. Amber is a unique form of fossilization (Hsieh and Plotnick, 2020). Although amber is patchily distributed in space and time, it is still especially suitable for investigating the evolution of terrestrial parasites associated with arthropods due to preservation potential (De Baets and Littlewood, 2015; Leung, 2017; De Baets et al., 2021a; De Baets et al., 2021b; Leung, 2021; Poinar, 2021). The high diversity of mermithid nematodes during the mid-Cretaceous as shown here provides a glimpse into the structure of ancient parasitic nematode–host associations and their evolution over the past 100 million years.

All data are available in the main text and/or the supplementary materials.

1. 1. Choo HY
   2. Kaya HK
   3. Kim JB

   (1989)

   Agamermis Unka (Mermithidae) Parasitism of Nilaparvata Lugens in rice fields in Korea

   Journal of Nematology 21:254–259..

   • PubMed
   • Google Scholar
2. 1. De Baets K
   2. Littlewood DTJ

   (2015) The importance of fossils in understanding the evolution of parasites and their vectors

   Advances in Parasitology 90:1–51.

   https://doi.org/10.1016/bs.apar.2015.07.001

   • PubMed
   • Google Scholar
3. Book

   1. De Baets K
   2. Dentzien-Dias P
   3. Harrison GWM
   4. Littlewood DTJ
   5. Parry LA

   (2021a) Fossil constraints on the timescale of parasitic helminth evolution

   In: De Baets K, Huntley JW, editors. The Evolution and Fossil Record of Parasitism: Identification and Macroevolution of Parasites. Cham, Switzerland: Springer International Publishing. pp. 231–271.

   https://doi.org/10.1007/978-3-030-42484-8

   • Google Scholar
4. Book

   1. De Baets K
   2. Huntley JW
   3. Klompmaker AA
   4. Schiffbauer JD
   5. Muscente AD

   (2021b) The fossil record of parasitism: its extent and taphonomic constraints

   In: De Baets K, Huntley JW, editors. The Evolution and Fossil Record of Parasitism: Coevolution and Paleoparasitological Techniques. Cham, Switzerland: Springer International Publishing. pp. 1–50.

   https://doi.org/10.1007/978-3-030-52233-9

   • Google Scholar
5. 1. Grimaldi DA
   2. Engel MS
   3. Nascimbene PC

   (2002) Fossiliferous Cretaceous amber from Myanmar (Burma): its rediscovery, biotic diversity, and paleontological significance

   American Museum Novitates 3361:1–71.

   https://doi.org/10.1206/0003-0082(2002)361<0001:FCAFMB>2.0.CO;2

   • Google Scholar
6. 1. Helden AJ

   (2008) First extant records of mermithid nematode parasitism of Auchenorrhyncha in Europe

   Journal of Invertebrate Pathology 99:351–353.

   https://doi.org/10.1016/j.jip.2008.05.005

   • PubMed
   • Google Scholar
7. 1. Hsieh S
   2. Plotnick RE

   (2020) The representation of animal behaviour in the fossil record

   Animal Behaviour 169:65–80.

   https://doi.org/10.1016/j.anbehav.2020.09.010

   • Google Scholar
8. Book

   1. International Commission on Zoological Nomenclature

   (1999)

   International Code of Zoological Nomenclature (4th ed)

   London: International Trust for Zoological Nomenclature.

   • Google Scholar
9. 1. Labandeira CC
   2. Sepkoski JJ

   (1993) Insect diversity in the fossil record

   Science 261:310–315.

   https://doi.org/10.1126/science.11536548

   • PubMed
   • Google Scholar
10. Book

    1. Labandeira CC

    (2005)

    Fossil history and evolutionary ecology of Diptera and their associations with plants

    In: Yeates DK, Wiegmann BM, editors. The Evolutionary Biology of Flies. New York: Columbia University Press. pp. 217–273.

    • Google Scholar
11. 1. Leung TLF

    (2017) Fossils of parasites: what can the fossil record tell us about the evolution of parasitism

    Biological Reviews 92:410–430.

    https://doi.org/10.1111/brv.12238

    • Google Scholar
12. Book

    1. Leung TLF

    (2021) Parasites of fossil vertebrates: what we know and what can we expect from the fossil record?

    In: De Baets K, Huntley JW, editors. The Evolution and Fossil Record of Parasitism: Identification and Macroevolution of Parasites. Cham: Springer International Publishing. pp. 1–28.

    https://doi.org/10.1007/978-3-030-42484-8

    • Google Scholar
13. Book

    1. Lorenzen S

    (1994)

    The Phylogenetic Systematics of Freeliving Nematodes

    The Ray Society.

    • Google Scholar
14. 1. Misof B
    2. Liu S
    3. Meusemann K
    4. Peters RS
    5. Donath A
    6. Mayer C
    7. Frandsen PB
    8. Ware J
    9. Flouri T
    10. Beutel RG
    11. Niehuis O
    12. Petersen M
    13. Izquierdo-Carrasco F
    14. Wappler T
    15. Rust J
    16. Aberer AJ
    17. Aspöck U
    18. Aspöck H
    19. Bartel D
    20. Blanke A
    21. Berger S
    22. Böhm A
    23. Buckley TR
    24. Calcott B
    25. Chen J
    26. Friedrich F
    27. Fukui M
    28. Fujita M
    29. Greve C
    30. Grobe P
    31. Gu S
    32. Huang Y
    33. Jermiin LS
    34. Kawahara AY
    35. Krogmann L
    36. Kubiak M
    37. Lanfear R
    38. Letsch H
    39. Li Y
    40. Li Z
    41. Li J
    42. Lu H
    43. Machida R
    44. Mashimo Y
    45. Kapli P
    46. McKenna DD
    47. Meng G
    48. Nakagaki Y
    49. Navarrete-Heredia JL
    50. Ott M
    51. Ou Y
    52. Pass G
    53. Podsiadlowski L
    54. Pohl H
    55. von Reumont BM
    56. Schütte K
    57. Sekiya K
    58. Shimizu S
    59. Slipinski A
    60. Stamatakis A
    61. Song W
    62. Su X
    63. Szucsich NU
    64. Tan M
    65. Tan X
    66. Tang M
    67. Tang J
    68. Timelthaler G
    69. Tomizuka S
    70. Trautwein M
    71. Tong X
    72. Uchifune T
    73. Walzl MG
    74. Wiegmann BM
    75. Wilbrandt J
    76. Wipfler B
    77. Wong TKF
    78. Wu Q
    79. Wu G
    80. Xie Y
    81. Yang S
    82. Yang Q
    83. Yeates DK
    84. Yoshizawa K
    85. Zhang Q
    86. Zhang R
    87. Zhang W
    88. Zhang Y
    89. Zhao J
    90. Zhou C
    91. Zhou L
    92. Ziesmann T
    93. Zou S
    94. Li Y
    95. Xu X
    96. Zhang Y
    97. Yang H
    98. Wang J
    99. Wang J
    100. Kjer KM
    101. Zhou X

    (2014) Phylogenomics resolves the timing and pattern of insect evolution

    Science 346:763–767.

    https://doi.org/10.1126/science.1257570

    • PubMed
    • Google Scholar
15. 1. Nickle WR

    (1972)

    A contribution to our knowledge of the Mermithidae (Nematoda)

    Journal of Nematology 4:113–146..

    • PubMed
    • Google Scholar
16. 1. Parry LA
    2. Boggiani PC
    3. Condon DJ
    4. Garwood RJ
    5. Leme JM
    6. McIlroy D
    7. Brasier MD
    8. Trindade R
    9. Campanha GAC
    10. Pacheco MLAF
    11. Diniz CQC
    12. Liu AG

    (2017) Ichnological evidence for meiofaunal bilaterians from the terminal Ediacaran and earliest Cambrian of Brazil

    Nature Ecology & Evolution 1:1455–1464.

    https://doi.org/10.1038/s41559-017-0301-9

    • Google Scholar
17. 1. Peters RS
    2. Krogmann L
    3. Mayer C
    4. Donath A
    5. Gunkel S
    6. Meusemann K
    7. Kozlov A
    8. Podsiadlowski L
    9. Petersen M
    10. Lanfear R
    11. Diez PA
    12. Heraty J
    13. Kjer KM
    14. Klopfstein S
    15. Meier R
    16. Polidori C
    17. Schmitt T
    18. Liu S
    19. Zhou X
    20. Wappler T
    21. Rust J
    22. Misof B
    23. Niehuis O

    (2017) Evolutionary history of the Hymenoptera

    Current Biology 27:1013–1018.

    https://doi.org/10.1016/j.cub.2017.01.027

    • PubMed
    • Google Scholar
18. Book

    1. Petersen JJ

    (1985) Nematodes as biological control agents: Part I. Mermithidae

    In: Baker JR, Muller R, editors. Advances in Parasitology. Academic Press. pp. 307–344.

    https://doi.org/10.1016/s0065-308x(08)60565-5

    • PubMed
    • Google Scholar
19. 1. Poinar GO
    2. Otieno WA

    (1974) Evidence of four Molts in the Mermithidae

    Nematologica 20:370.

    https://doi.org/10.1163/187529274X00456

    • Google Scholar
20. Book

    1. Poinar GO

    (1975)

    Entomogenous Nematodes: A Manual and Host List of Insect-Nematode Associations

    Leiden: E. J. Brill.

    • Google Scholar
21. Book

    1. Poinar GO

    (1979)

    Nematodes for Biological Control of Insects (1st ed)

    Boca Raton: CRC Press.

    • Google Scholar
22. Book

    1. Poinar GO

    (1983)

    The Natural History of Nematodes

    Englewood Cliffs: Prentice Hall.

    • Google Scholar
23. 1. Poinar GO
    2. Acra A
    3. Acra F

    (1994)

    Earliest fossil nematode (Mermithidae) in Cretaceous Lebanese amber

    Fundamental and Applied Nematology 17:475–477.

    • Google Scholar
24. 1. Poinar GO

    (2001a) Heydenius brownii sp. n. (Nematoda: Mermithidae) parasitising a planthopper (Homoptera: Achilidae) in Baltic amber

    Nematology 3:753–757.

    https://doi.org/10.1163/156854101753625263

    • Google Scholar
25. Book

    1. Poinar GO

    (2001b) Nematoda and Nematomorpha

    In: Thorp JH, Covich AP, editors. Ecology and Classification of North American Freshwater Invertebrates. New York: Academic Press. pp. 255–295.

    https://doi.org/10.1016/B978-0-12-374855-3.00009-1

    • Google Scholar
26. 1. Poinar GO
    2. Buckley R

    (2006) Nematode (Nematoda: Mermithidae) and hairworm (Nematomorpha: Chordodidae) parasites in early Cretaceous amber

    Journal of Invertebrate Pathology 93:36–41.

    https://doi.org/10.1016/j.jip.2006.04.006

    • Google Scholar
27. 1. Poinar GO
    2. Kerp H
    3. Hass H

    (2008) Palaeonema phyticum gen. n., sp. n. (Nematoda: Palaeonematidae fam. n.), a Devonian nematode associated with early land plants

    Nematology 10:9–14.

    https://doi.org/10.1163/156854108783360159

    • Google Scholar
28. 1. Poinar GO
    2. Sarto i Monteys V

    (2008) Mermithids (Nematoda: Mermithidae) of biting midges (Diptera: Ceratopogonidae): Heleidomermis cataloniensis n. sp. from Culicoides circumscriptus Kieffer in Spain and a species of Cretacimermis Poinar, 2001 from a ceratopogonid in Burmese amber

    Systematic Parasitology 69:13–21.

    https://doi.org/10.1007/s11230-007-9091-9

    • PubMed
    • Google Scholar
29. Book

    1. Poinar GO

    (2011) The Evolutionary History of Nematodes: As Revealed in Stone, Amber and Mummies

    Brill, Amersfoort.

    https://doi.org/10.1163/9789047428664

    • Google Scholar
30. Book

    1. Poinar GO

    (2015a) Phylum Nemata

    In: Thorp JH, Rogers DC, editors. Thorp and Covich’s Freshwater Invertebrates (Fourth Edition). Boston: Academic Press. pp. 273–302.

    https://doi.org/10.1016/B978-0-12-385026-3.00014-0

    • Google Scholar
31. 1. Poinar GO

    (2015b) The geological record of parasitic nematode evolution

    Advances in Parasitology 90:53–92.

    https://doi.org/10.1016/bs.apar.2015.03.002

    • PubMed
    • Google Scholar
32. 1. Poinar GO

    (2017) A mermithid nematode, Cretacimermis aphidophilus sp. n. (Nematoda: Mermithidae), parasitising an aphid (Hemiptera: Burmitaphididae) in Myanmar amber: a 100 million year association

    Nematology 19:509–513.

    https://doi.org/10.1163/15685411-00003063

    • Google Scholar
33. Book

    1. Poinar GO

    (2021) Fossil record of viruses, parasitic bacteria and parasitic protozoa

    In: De Baets K, Huntley JW, editors. The Evolution and Fossil Record of Parasitism: Identification and Macroevolution of Parasites. Cham: Springer International Publishing. pp. 29–68.

    https://doi.org/10.1007/978-3-030-42484-8

    • Google Scholar
34. 1. Ross AJ

    (2023) Supplement to the Burmese (Myanmar) amber checklist and bibliography, 2022

    Palaeoentomology 6:22–40.

    https://doi.org/10.11646/palaeoentomology.6.1.6

    • Google Scholar
35. 1. Shi G
    2. Grimaldi DA
    3. Harlow GE
    4. Wang J
    5. Wang J
    6. Yang M
    7. Lei W
    8. Li Q
    9. Li X

    (2012) Age constraint on Burmese amber based on U–PB dating of zircons

    Cretaceous Research 37:155–163.

    https://doi.org/10.1016/j.cretres.2012.03.014

    • Google Scholar
36. 1. Sohn JC
    2. Labandeira CC
    3. Davis DR

    (2015) The fossil record and taphonomy of butterflies and moths (Insecta, Lepidoptera): implications for evolutionary diversity and divergence-time estimates

    BMC Evolutionary Biology 15:12.

    https://doi.org/10.1186/s12862-015-0290-8

    • Google Scholar
37. 1. Stoffolano JG

    (1973) Host specificity of entomophilic nematodes—A review

    Experimental Parasitology 33:263–284.

    https://doi.org/10.1016/0014-4894(73)90033-7

    • Google Scholar
38. 1. Szwedo J
    2. Wang B
    3. Soszynska-Maj A
    4. Azar D
    5. Ross A

    (2020) International Palaeoentomological Society Statement

    Palaeoentomology 3:221–222.

    https://doi.org/10.11646/palaeoentomology.3.3.1

    • Google Scholar
39. 1. Thomas JA
    2. Frandsen PB
    3. Prendini E
    4. Zhou X
    5. Holzenthal RW

    (2020) A multigene phylogeny and timeline for Trichoptera (Insecta)

    Systematic Entomology 45:670–686.

    https://doi.org/10.1111/syen.12422

    • Google Scholar
40. Book

    1. Thu K
    2. Zaw K

    (2017) Chapter 23 Gem deposits of Myanmar

    In: Barber AJ, Zaw K, Crow MJ, editors. Myanmar: Geology, Resources and Tectonics. The Geological Society. pp. 497–529.

    https://doi.org/10.1144/M48.23

    • Google Scholar
41. 1. van den Hoogen J
    2. Geisen S
    3. Routh D
    4. Ferris H
    5. Traunspurger W
    6. Wardle DA
    7. de Goede RGM
    8. Adams BJ
    9. Ahmad W
    10. Andriuzzi WS
    11. Bardgett RD
    12. Bonkowski M
    13. Campos-Herrera R
    14. Cares JE
    15. Caruso T
    16. de Brito Caixeta L
    17. Chen X
    18. Costa SR
    19. Creamer R
    20. Mauro da Cunha Castro J
    21. Dam M
    22. Djigal D
    23. Escuer M
    24. Griffiths BS
    25. Gutiérrez C
    26. Hohberg K
    27. Kalinkina D
    28. Kardol P
    29. Kergunteuil A
    30. Korthals G
    31. Krashevska V
    32. Kudrin AA
    33. Li Q
    34. Liang W
    35. Magilton M
    36. Marais M
    37. Martín JAR
    38. Matveeva E
    39. Mayad EH
    40. Mulder C
    41. Mullin P
    42. Neilson R
    43. Nguyen TAD
    44. Nielsen UN
    45. Okada H
    46. Rius JEP
    47. Pan K
    48. Peneva V
    49. Pellissier L
    50. Carlos Pereira da Silva J
    51. Pitteloud C
    52. Powers TO
    53. Powers K
    54. Quist CW
    55. Rasmann S
    56. Moreno SS
    57. Scheu S
    58. Setälä H
    59. Sushchuk A
    60. Tiunov AV
    61. Trap J
    62. van der Putten W
    63. Vestergård M
    64. Villenave C
    65. Waeyenberge L
    66. Wall DH
    67. Wilschut R
    68. Wright DG
    69. Yang J
    70. Crowther TW

    (2019) Soil nematode abundance and functional group composition at a global scale

    Nature 572:194–198.

    https://doi.org/10.1038/s41586-019-1418-6

    • Google Scholar
42. 1. Wang B
    2. Xu C
    3. Jarzembowski EA

    (2022) Ecological radiations of insects in the Mesozoic

    Trends in Ecology & Evolution 37:529–540.

    https://doi.org/10.1016/j.tree.2022.02.007

    • Google Scholar
43. Book

    1. Yeates GW
    2. Ferris H
    3. Moens T
    4. Van Der Putten WH

    (2009) The role of nematodes in ecosystems

    In: Wilson MJ, Kakouli-Duarte T, editors. Nematodes as Environmental Indicators. CAB International. pp. 1–44.

    https://doi.org/10.1079/9781845933852.0000

    • Google Scholar
44. 1. Yu T
    2. Thomson U
    3. Mu L
    4. Ross A
    5. Kennedy J
    6. Broly P
    7. Xia F
    8. Zhang H
    9. Wang B
    10. Dilcher D

    (2019) An ammonite trapped in Burmese amber

    PNAS 116:11345–11350.

    https://doi.org/10.1073/pnas.1821292116

    • PubMed
    • Google Scholar
45. 1. Zhang SQ
    2. Che LH
    3. Li Y
    4. Pang H
    5. Ślipiński A
    6. Zhang P

    (2018) Evolutionary history of Coleoptera revealed by extensive sampling of genes and species

    Nature Communications 9:205.

    https://doi.org/10.1038/s41467-017-02644-4

    • PubMed
    • Google Scholar
46. 1. Zhang Y
    2. Li S
    3. Li H
    4. Wang R
    5. Zhang KQ
    6. Xu J

    (2020) Fungi-nematode interactions: diversity, ecology, and biocontrol prospects in agriculture

    Journal of Fungi 6:206.

    https://doi.org/10.3390/jof6040206

    • PubMed
    • Google Scholar

Decision letter after peer review:

Thank you for submitting your article "Widespread mermithid nematode parasitism of Cretaceous insects" for consideration by eLife. Your article has been reviewed by 3 peer reviewers including David Marjanović as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by George Perry as the Senior Editor.

Two of the reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) The abstract mentions statistical analyses, but only pie charts are presented. The data you present in the supplementary material could easily, and should, be used for a statistical analysis to test your hypothesis of a shift toward holometabolous insects as hosts. Please also briefly address, in the text, the possibility of collection bias: are, for example, beetles underrepresented in museum collections of insects in amber, and are such biases likely to be identical for all sources of amber (Baltic amber has been sampled for centuries, Kachin amber only for a decade or two)?

2) Please write for a slightly wider audience. For example, please explain how trophosomes are recognized, and how fossil mermithids can be differentiated from, for example, nematomorphs; please also mention if mermithids are known to exit their hosts as a panic reaction. If you can illustrate and describe the exit wounds, this would greatly strengthen the manuscript against the possibility that some of the associations might be accidental.

3) The nomenclature needs a few minor improvements. I also urge you to take this opportunity to publish the genus name Cretacimermis properly, because that has not been done so far – the original publication lacks a diagnosis, which is required even for collective groups, so the name "Cretacimermis" is not currently available and does not even compete for homonymy.

Reviewer #1 (Recommendations for the authors):

I am not familiar with nematodes or amber, so I only wrote my review after receiving and reading the other reviews, and I have focused on the topics I am more competent in.

Comments on science

Given the wide readership of eLife, it would be good to explicitly state the evidence that these rather featureless elongate fossils represent specifically the parasitic stage of, specifically, mermithids (and not for example nematomorphs or a whole new group of nematodes). In your descriptions you mention trophosomes (implying parasitism) and details of the cuticle; is any of this diagnostic for Mermithidae, or is something else that you can see? How are trophosomes recognized? (I see the brown fragments you point out in Figure 4H; why do you interpret them as a trophosome?) Is it possible to illustrate or at least describe the exit wounds in more detail in the specimens where the mermithids have fully exited?

Can you exclude collection bias as a factor in the apparent shift to holometabolous hosts?

Line 25: By "statistical analyses", do you mean the pie charts in Figure 9? That's a presentation of data, not an analysis. The term made me expect mathematics.

64: Are mermithids known to exit their hosts as a flight reaction when the host is stressed?

Comments on nomenclature

I was wrong in my pre-review: collective groups are explicitly allowed under the ICZN; and diagnoses do not need to be limited to morphological characters, they can refer to behavior. However, collective species are not allowed – only collective genera and subgenera (ICZN Articles 10.3, 42.2.1). The species names you propose are therefore fine, just don't call them "collective".

However, because genus names must be published with a diagnosis even if they're intended for a collective genus, "Cretacimermis" is actually unavailable: for purposes of zoological nomenclature, this name does not exist. I quote the original publication (Poinar 2001b: 262): "Since the generic placement of this specimen was based in part on its host family, it is now prudent to transfer the species libani from Heleidomermis Rubstov into a new genus Cretacimermis Poinar. Thus, this nematode, which is the oldest definite fossil nematode, should now be called Cretacimermis libani Poinar, Acra and Acra (1994)." This is not "a description or definition that states in words characters that are purported to differentiate the taxon" (ICZN Art. 3.1.1). I therefore strongly recommend that you validly publish Cretacimermis here as a new name. (Interestingly, you don't need to select a type species, because collective groups do not have type species: ICZN Art. 13.3.2, 42.3.1, 66, 67.14. This must be precisely because species cannot be collective groups.)

I also strongly recommend against using times or places in diagnoses (if I find, say, a mermithid exiting an archaeognath in Late Cretaceous amber from New Jersey, do I really have to erect a new species name for it instead of referring it to "Cretacimermis" incredibilis…?), let alone the type of preservation (amber), but this is not forbidden by the ICZN. ("Character", as used in Art. 3.1.1, is defined in the Glossary as: "Any attribute of organisms used for recognizing, differentiating, or classifying taxa.")

Line 101: Why "extraordinary" and not simply "incredible"?

129: The species name itself is fine, but adelphos means "brother", not "sister"; "sister" is "adelphê".

144: Much simpler: "The species epithet is the Latin 'directa' = arranged in a straight line."

172, 175, 193, 225: Another reviewer has pointed out that not all bugs are bedbugs, and that naming a planthopper parasite after bedbugs may therefore not be a good idea. Given that you name "C." manicapsoci after the type genus of Manicapsocidae (not directly after Manicapsocidae) and likewise "C." cecidomyiae after they type genus of Cecidomyiidae, you could also name the planthopper parasite "C. perforissi", after the type genus of Perforissidae.

226: Being a valid name, Cecidomyia should be preceded by a comma rather than surrounded by quotation marks. You can also delete it entirely; you didn't spell out Manicapsocus in line 193.

286: Replace "Archeognatha" by "Archaeognatha".

365: Is he Peter or Petr?

593-594 and Supplementary Table: As you correctly state in the text, the authors of "C." chironomae are not Poinar, 2011, but Grimaldi et al., 2002.

Comments on style and language

Line 27-28: "have […] exploited" is from the point of view of the present, so it clashes with "until the Cenozoic"; replace it by "had […] exploited".

34: Replace "one" by "some" to fit the plural "Nematodes […] are" in the preceding line.

40: Likewise, replace "a plant parasite" by "plant parasites" to stay in the plural.

41: Replace "can" by "could", "may" or "might".

63: It took me a second to understand "invertebrate parasitic habits"; in this form it would mean that their habits are both parasitic and invertebrate. Please use a hyphen in "invertebrate-parasitic habits" (or reword entirely, e.g. "parasitism of invertebrates").

292: Replace "be" by "have been" simply because the mid-Cretaceous is in the past.

294: Remove the comma; it wrongly implies that H. brownii is the only mermithid you're talking about here (and would need to be matched by another comma after the name).

313-315: "depicted" refers to literal pictures; replace it by "shown", "recorded" or "demonstrated". More importantly, however, the entire clause seems like it was incompletely edited; in its current form it states that mermithids parasitized dipterans before they parasitized anything else. I think you mean: "also, the first fossil animal that was found to host a mermithid is a dipteran from Early Cretaceous Lebanese amber (Poinar et al., 1994)" or something to that effect.

316: Replace "developed" by "develop".

325: Replace "be built" by "have formed".

Figure 8: Replace "~" by "-" (Ctrl+minus on the numeric block in MS Word, Alt+0150 on the numeric block in other software); "~" will not be understood as "to" outside of China – it will be mistaken as having its mathematical meaning of "about".

Reviewer #2 (Recommendations for the authors):

Overall, I would highly recommend this manuscript be accepted for publication, however, I do have a few minor comments about the manuscript that I would like the authors to address before the manuscript can be accepted for publication.

L62: "their large size" should be changed to "their relatively large size" since while it is true that mermithids are fairly large in terms of nematodes, there are also many nematodes which reach much, much larger sizes than mermithids.

L175: I question naming this species after bedbugs (Cimex) since the host was a planthopper. Wouldn't it be more appropriate to name the species after the host taxa?

Reviewer #3 (Recommendations for the authors):

As stated before this monumental description and valuable new data on fossil parasitic parasites is timely and very welcome. I just feel you could your own observations and work a bit short by not including a supporting statistical analysis of the shift in host exploitation (at minimum an analysis with error or confidence intervals would be appropriate). You alluded to a statistical analysis in the abstract but I could not find it in the manuscript. I feel this can be easily achieved with the new literature and data you compiled and would make your interpretations much more robust and fundamental. Here are my main suggestions on how you may achieve that goal:

Line 40-41: the statements need to be backed up by more recent publications – the authors could cite De Baets et al. 2021a in this context which reviewed the nematode fossil record and the authors already cite. In addition, the authors could cite Parry et al. (2017) who most recently reported sinusoidal traces which might reflect nematode activity but also cautioned for possible alternative interpretations.

Line 59: the fossils are also treated as parasitic nematodes in another publication in the same volumes by De Baets et al. (2021b); this chapter might be of interest as the authors also depict the nematode fossil record through the Phanerozoic and clearly demonstrate that amber provides one of the richest records of nematode body fossils including parasitic ones.

Line 76-79: Indeed, also the changes in proportion for heterometabolous to holometabolous insects could be mentioned. I feel that you undersell the importance of your study by dumping this information in the supplementary material. You also allude to a statistical analysis in the abstract but as far as I can see you are just naming percentages and have pie diagrams which can be widely deceiving (e.g., Rougier et al. 2014). Depicting the changing proportions of parasitic nematodes from heterometabolous to holometabolous insects would make your study easier to follow and reproduce as well as your interpretations more convincing.

Lines 100, 114, 128, 143, 157, 174, 192, 210, 224, Below each new species, the zoobank number should be added.

Line 300: You need to back up this statement with a reference. De Baets et al. 2021 (not to be confused with the other reference you already cite) recently depicted the known record of nematode body fossils and show that amber is one of its richest sources of nematodes backing up your statement.

Line 302-305: This is an important result and therefore the information should not be buried in the supplementary material. It is crucial to analyze/depict this information in a bar plot rather than pie diagrams which although graphically appealing might distort information (compare Figure 1.3 in De Baets et al. 2021) and I feel this could be done by separating those species in heterometabolous and holometabolous insects per amber deposit. To make your interpretations more robust and deal with differences in sampling heterometabolous versus holometabolous insects, at minimum binomial error or confidence intervals should be added to this plot (Raup 1991; Takeda and Tanabe 2014) if not more broadsweeping statistical analyses. Confidence intervals are more conservative and can easily be calculated in Matlab with Binofit function or R with the binom.confint function of the package Binom as well as other software. I feel such analyses could be done for both mermithids and all (parasitic) nematodes in those 3 amber deposits. Particularly tabulated data and depiction of the distribution of mermithids in modern insects would be helpful to understand the context. This might be trivial to you but not so to the reader who does not necessarily has access to all relevant literature.

Line 330-331: In addition to De Baets et al. 2021a, 2021b would be highly relevant in this context.

Line 344-353: I appreciate this explicit ethics statement and I feel it should be mandatory in all kind of studies dealing with fossils not just amber.

Line 390: Thank you for providing the raw data but I feel your study would also benefit from depicting this data in a bar plot with confidence intervals (as explained before) as well as add data and host distributions of modern mermithids.

Suggested references:

De Baets, K., Dentzien-Dias, P., Harrison, G.W.M., Littlewood, D.T.J., Parry, L.A. (2021a). Fossil Constraints on the Timescale of Parasitic Helminth Evolution. In: De Baets, K., Huntley, J.W. (eds) The Evolution and Fossil Record of Parasitism. Topics in Geobiology 49: 231-271. Springer, Cham. (already cited)

De Baets, K., Huntley, J.W., Klompmaker, A.A., Schiffbauer, J.D., Muscente, A.D. (2021b). The Fossil Record of Parasitism: Its Extent and Taphonomic Constraints. In: De Baets, K., Huntley, J.W. (eds) The Evolution and Fossil Record of Parasitism. Topics in Geobiology 50: 1-50. Springer, Cham.

Parry, L. A., Boggiani, P. C., Condon, D. J., Garwood, R. J., Leme, J. D. M., McIlroy, D., … & Liu, A. G. (2017). Ichnological evidence for meiofaunal bilaterians from the terminal Ediacaran and earliest Cambrian of Brazil. Nature Ecology & Evolution, 1(10), 1455-1464.

Raup, D. M. (1991). The future of analytical paleobiology. Short courses in paleontology, 4, 207-216.

Rougier, N. P., Droettboom, M., & Bourne, P. E. (2014). Ten simple rules for better figures. PLoS computational biology, 10(9), e1003833.

Takeda, Y., & Tanabe, K. (2014). Low durophagous predation on Toarcian (Early Jurassic) ammonoids in the northwestern Panthalassa shelf basin. Acta Palaeontologica Polonica, 60(4), 781-794.

Essential revisions:

1) The abstract mentions statistical analyses, but only pie charts are presented. The data you present in the supplementary material could easily, and should, be used for a statistical analysis to test your hypothesis of a shift toward holometabolous insects as hosts. Please also briefly address, in the text, the possibility of collection bias: are, for example, beetles underrepresented in museum collections of insects in amber, and are such biases likely to be identical for all sources of amber (Baltic amber has been sampled for centuries, Kachin amber only for a decade or two)?

Thanks. We fully agreed with the reviewer and revised this part. We have added a new Figure 9B and Table 1 to our paper. Indeed, collection bias is almost present in all amber biotas. However, we believe we have robust reasons to argue that the shift to holometabolous hosts does exist. Although Kachin amber has only been studied extensively in the last two decades (compared with centuries of study in Baltic amber or Dominican amber), it has become by far the most intensively studied amber biota since its Cretaceous age was appreciated, now comprising an exceptional 700 families (Ross, 2023). Also, the fossil record of holometabolous insects is clearly much better than non-holometabolous insects in Kachin amber (1296 spp. vs 465 spp. respectively). But as shown in our paper, the nematodes we found in Kachin amber are mainly associated with heterometabolous insects. Therefore, even if collection bias might exist, such as the presence of some unreported nematode-Holometabola associations, we believe our conclusion about the shift is robust. We also add some explanation in our paper, please see line 350–356 in the clean copy of our revised manuscript.

2) Please write for a slightly wider audience. For example, please explain how trophosomes are recognized, and how fossil mermithids can be differentiated from, for example, nematomorphs; please also mention if mermithids are known to exit their hosts as a panic reaction. If you can illustrate and describe the exit wounds, this would greatly strengthen the manuscript against the possibility that some of the associations might be accidental.

Thank you very much for pointing out this issue. Please see below.

3) The nomenclature needs a few minor improvements. I also urge you to take this opportunity to publish the genus name Cretacimermis properly, because that has not been done so far – the original publication lacks a diagnosis, which is required even for collective groups, so the name "Cretacimermis" is not currently available and does not even compete for homonymy.

Thanks. We formally erect this genus in this paper.

Reviewer #1 (Recommendations for the authors):

I am not familiar with nematodes or amber, so I only wrote my review after receiving and reading the other reviews (I am the Reviewing Editor), and I have focused on the topics I am more competent in.

Comments on science

Given the wide readership of eLife, it would be good to explicitly state the evidence that these rather featureless elongate fossils represent specifically the parasitic stage of, specifically, mermithids (and not for example nematomorphs or a whole new group of nematodes). In your descriptions you mention trophosomes (implying parasitism) and details of the cuticle; is any of this diagnostic for Mermithidae, or is something else that you can see? How are trophosomes recognized? (I see the brown fragments you point out in Figure 4H; why do you interpret them as a trophosome?) Is it possible to illustrate or at least describe the exit wounds in more detail in the specimens where the mermithids have fully exited?

Thank you very much for pointing out these issues. First, the parasitic stage of fossil mermithids is almost impossible to determine due to the lack of detailed structure. In fact, it is also very difficult to determine the stage of extant mermithids only based on morphological characters. It is a pity that the number of moults is based on experimental observation (Poinar, 1974), which is impossible to duplicate in fossil research.

The identification of fossil mermithids is based mainly on their relatively large size, the morphological comparison with extant mermithids (body shape, length, body diameter, tail structure, etc.), and their position and posture in relation to the insects. Other invertebrateparasitic nematodes are usually relatively short, not coiled, and much smaller than their hosts, but mermithids are relatively long, coiled, and large compared with their hosts (Poinar, 2011). Nematomorphs are also long and big, but their epicuticles are normally crossed by grooves or furrows, leaving small elevations of irregular areas (areoles) between them (Poinar, 1999, 2001, 2006). We have therefore added some explanation at the beginning of Systematic palaeontology. Please see line 86-90 in the clean copy.

Trophosome are intestines of mermithids. As mermithids develop, the intestine becomes detached from the remainder of the alimentary tract and serves as a food storage organ (trophosome) (Poinar, 2015). Trophosomes can usually be detected in fossil mermithids by their different hues (Poinar, 2011). In order to make it readable for a wider audience, we have added some arrows and explanations in our figures.

Finally, exit wounds are hard to detect even in extant insects because when mermithids have left, these wounds tend to close up. For our specimens, we indeed have evidence (direct or indirect) to argue these associations are not accidental. We revised our figures and also have added these comments as “Remarks” in Systematic palaeontology, please see lines 137–139, 154–156, 171–172, 205–207, 225–227, 245–249, 263–264, 278–279, 298 in the clean copy.

Cretacimermis incredibilis: As we illustrated in Figure 2D, the tail end of the mermithids is adjacent to an exit wound on the host, indicating a true parasitic association.

Cretacimermis calypta: Although no exit wound can be clearly found, this mermithid is closely coiled around the dragonfly, indicating that the nematode was just emerging from the host.

Cretacimermis adelphe: The posterior part of the abdomen of this earwig has been damaged; it is most likely that these mermithids exited from the host through this wound.

Cretacimermis directa: There is no distinct wound on this cricket’s body, but the nematode is adjacent to it, and there is no other insect nearby. Therefore, it is most likely that the mermithid was just emerging from the host.

Cretacimermis longa: These two nematodes are still in the process of exiting. Also, the abdomen of the adult cockroach is empty and therefore probably contained the developing nematode.

Cretacimermis perforissi: The abdomen of the first perforissid planthopper is clearly empty and probably contained the developing nematode. The abdomen of the second perforissid planthopper is broken, which could be the result of the emerging mermithid.

Cretacimermis manicapsoci: first piece: there is no distinct wound on the barklouse’s body, but the nematode is adjacent to it, and there is no other insect big enough nearby. Therefore, it is most likely that the mermithid was just emerging from the host. Second piece: the abdomen of the second perforissid planthopper is lost, which could be the result of the emerging mermithid.

Cretacimermis psoci: The abdomen of the barklouse is broken, which could be the result of the emerging of mermithid.

References:

Poinar GO, Otieno WA. 1974. Evidence of four molts in the Mermithidae. Nematologica 20:370. DOI: https://doi.org/10.1163/187529274X00456

Poinar GO, 1999. Paleochordodes protus n.g., n.sp. (Nematomorpha, Chordodidae), parasites of a fossil cockroach, with a critical examination of other fossil hairworms and helminths of extant cockroaches (Insecta: Blattaria). Invertebrate Biology 118:109-115. DOI: https://doi.org/10.2307/3227053

Poinar GO, 2001. Nematoda and Nematomorpha, in: Thorp JH, Covich AP (Eds.), Ecology and classification of North American freshwater invertebrates, Second ed. Academic Press, New York, pp. 255–295.

Poinar GO, Buckley R. 2006. Nematode (Nematoda: Mermithidae) and hairworm (Nematomorpha: Chordodidae) parasites in Early Cretaceous amber. Journal of Invertebrate Pathology 93:36–41. DOI: https://doi.org/10.1016/j.jip.2006.04.006

Poinar GO, 2011. The evolutionary history of nematodes: as revealed in stone, amber and mummies. Brill, Amersfoort, the Netherlands.

Poinar GO, 2015. Chapter 14 – Phylum Nemata, in: Thorp JH, Rogers DC (Eds.), Thorp and Covich's Freshwater invertebrates (Fourth Edition). Academic Press, Boston, pp. 273–302.

Can you exclude collection bias as a factor in the apparent shift to holometabolous hosts?

Thanks. The collection bias does not affect our result. Please see Essential revision #2.

Line 25: By "statistical analyses", do you mean the pie charts in Figure 9? That's a presentation of data, not an analysis. The term made me expect mathematics.

Thanks. We calculated the 95% CI using the Agresti-Coull method of the “binom.confint” function from the binom R package (https://cran.r-project.org/package=binom) of R 4.2.2. We also added a new Figure 9B and Table 1 to our paper.

64: Are mermithids known to exit their hosts as a flight reaction when the host is stressed?

Thank you. Yes, nematodes tend to leave their hosts when their hosts are threated, e.g., caught by resin flow. That is why many fossil nematodes that occur in amber are very close to their hosts or just emerging from their hosts. We also add some words to explain this. Please see lines 53–56 in the clean copy.

Comments on nomenclature

I was wrong in my pre-review: collective groups are explicitly allowed under the ICZN; and diagnoses do not need to be limited to morphological characters, they can refer to behavior. However, collective species are not allowed – only collective genera and subgenera (ICZN Articles 10.3, 42.2.1). The species names you propose are therefore fine, just don't call them "collective".

Thanks. We deleted all the “Collective species” before our new species.

However, because genus names must be published with a diagnosis even if they're intended for a collective genus, "Cretacimermis" is actually unavailable: for purposes of zoological nomenclature, this name does not exist. I quote the original publication (Poinar 2001b: 262): "Since the generic placement of this specimen was based in part on its host family, it is now prudent to transfer the species libani from Heleidomermis Rubstov into a new genus Cretacimermis Poinar. Thus, this nematode, which is the oldest definite fossil nematode, should now be called Cretacimermis libani Poinar, Acra and Acra (1994)." This is not "a description or definition that states in words characters that are purported to differentiate the taxon" (ICZN Art. 3.1.1). I therefore strongly recommend that you validly publish Cretacimermis here as a new name. (Interestingly, you don't need to select a type species, because collective groups do not have type species: ICZN Art. 13.3.2, 42.3.1, 66, 67.14. This must be precisely because species cannot be collective groups.)

Thank you very much for pointing out this issue. We formally erect this genus in this paper. Collectives do not contain type species because they are not natural genera. Please see lines 104–123 in the clean copy.

I also strongly recommend against using times or places in diagnoses (if I find, say, a mermithid exiting an archaeognath in Late Cretaceous amber from New Jersey, do I really have to erect a new species name for it instead of referring it to "Cretacimermis" incredibilis…?), let alone the type of preservation (amber), but this is not forbidden by the ICZN. ("Character", as used in Art. 3.1.1, is defined in the Glossary as: "Any attribute of organisms used for recognizing, differentiating, or classifying taxa.")

Thanks. We also do not want to simply use times or places in diagnoses; however, it is almost impossible to identify fossil nematodes at generic and/or specific level based on morphology and we therefore have to use some other information in diagnoses like times, places and hosts as valuable circumstantial evidence. The same method is widely used in fossil nematodes. For example, Heydenius Taylor, 1935 was erected as a collective genus for all fossil mermithids from the Cenozoic, and Heydenius neotropicus Poinar, 2011 is for those infecting Chironomidae from Dominican amber, Heydenius matutinus (Menge, 1866) Taylor, 1935 is for those infecting Chironomidae from Baltic amber. Thus, if we do find a mermithid exiting an archaeognathan in Late Cretaceous amber from New Jersey, a new species is reasonable as unlikely to have infected chironomids in Dominican amber. It is therefore a more natural solution.

Line 101: Why "extraordinary" and not simply "incredible"?

Thanks. Corrected.

129: The species name itself is fine, but adelphos means "brother", not "sister"; "sister" is "adelphê".

Thanks. Corrected. We are very grateful for your comments and suggestions about the nomenclature.

144: Much simpler: "The species epithet is the Latin 'directa' = arranged in a straight line."

Thanks. Corrected.

172, 175, 193, 225: Another reviewer has pointed out that not all bugs are bedbugs, and that naming a planthopper parasite after bedbugs may therefore not be a good idea. Given that you name "C." manicapsoci after the type genus of Manicapsocidae (not directly after Manicapsocidae) and likewise "C." cecidomyiae after they type genus of Cecidomyiidae, you could also name the planthopper parasite "C. perforissi", after the type genus of Perforissidae.

Thanks. Corrected.

226: Being a valid name, Cecidomyia should be preceded by a comma rather than surrounded by quotation marks. You can also delete it entirely; you didn't spell out Manicapsocus in line 193.

Thanks. Deleted.

286: Replace "Archeognatha" by "Archaeognatha".

Thanks. Corrected.

365: Is he Peter or Petr?

Thanks. He is Peter

593-594 and Supplementary Table: As you correctly state in the text, the authors of "C." chironomae are not Poinar, 2011, but Grimaldi et al., 2002.

Thanks. However, although Grimaldi et al., 2002 first reported two mermithid nematode parasites emerging from the abdomen of a female chironomid midge, they did not describe it. It was “Poinar, 2011” who formally described these nematodes and proposed this new species.

Comments on style and language

Line 27-28: "have […] exploited" is from the point of view of the present, so it clashes with "until the Cenozoic"; replace it by "had […] exploited".

Thanks. Corrected.

34: Replace "one" by "some" to fit the plural "Nematodes […] are" in the preceding line.

Thanks. Corrected.

40: Likewise, replace "a plant parasite" by "plant parasites" to stay in the plural.

Thanks. We replaced “fossils” by “fossil” since there is only one nematode known from Rhynie Chert so far.

41: Replace "can" by "could", "may" or "might".

Thanks. Corrected.

63: It took me a second to understand "invertebrate parasitic habits"; in this form it would mean that their habits are both parasitic and invertebrate. Please use a hyphen in "invertebrate-parasitic habits" (or reword entirely, e.g. "parasitism of invertebrates").

Thanks. Corrected.

292: Replace "be" by "have been" simply because the mid-Cretaceous is in the past.

Thanks. Corrected.

294: Remove the comma; it wrongly implies that H. brownii is the only mermithid you're talking about here (and would need to be matched by another comma after the name).

Thanks. Corrected.

313-315: "depicted" refers to literal pictures; replace it by "shown", "recorded" or "demonstrated". More importantly, however, the entire clause seems like it was incompletely edited; in its current form it states that mermithids parasitized dipterans before they parasitized anything else. I think you mean: "also, the first fossil animal that was found to host a mermithid is a dipteran from Early Cretaceous Lebanese amber (Poinar et al., 1994)" or something to that effect.

Thanks. Corrected.

316: Replace "developed" by "develop".

Thanks. Corrected.

325: Replace "be built" by "have formed".

Thanks. Corrected.

Figure 8: Replace "~" by "-" (Ctrl+minus on the numeric block in MS Word, Alt+0150 on the numeric block in other software); "~" will not be understood as "to" outside of China – it will be mistaken as having its mathematical meaning of "about".

Thanks. Corrected.

Reviewer #2 (Recommendations for the authors):

Overall, I would highly recommend this manuscript be accepted for publication, however, I do have a few minor comments about the manuscript that I would like the authors to address before the manuscript can be accepted for publication.

L62: "their large size" should be changed to "their relatively large size" since while it is true that mermithids are fairly large in terms of nematodes, there are also many nematodes which reach much, much larger sizes than mermithids.

Thanks. Corrected.

L175: I question naming this species after bedbugs (Cimex) since the host was a planthopper. Wouldn't it be more appropriate to name the species after the host taxa?

Thanks. We renamed this species as Cretacimermis perforissi based on the editor’s comments (please see comment 16).

Reviewer #3 (Recommendations for the authors):

As stated before this monumental description and valuable new data on fossil parasitic parasites is timely and very welcome. I just feel you could your own observations and work a bit short by not including a supporting statistical analysis of the shift in host exploitation (at minimum an analysis with error or confidence intervals would be appropriate). You alluded to a statistical analysis in the abstract but I could not find it in the manuscript. I feel this can be easily achieved with the new literature and data you compiled and would make your interpretations much more robust and fundamental. Here are my main suggestions on how you may achieve that goal:

Thanks. We have added a new Figure 9B and Table 1 to our paper.

Line 40-41: the statements need to be backed up by more recent publications – the authors could cite De Baets et al. 2021a in this context which reviewed the nematode fossil record and the authors already cite. In addition, the authors could cite Parry et al. (2017) who most recently reported sinusoidal traces which might reflect nematode activity but also cautioned for possible alternative interpretations.

Thanks. We have added these two references here.

Line 59: the fossils are also treated as parasitic nematodes in another publication in the same volumes by De Baets et al. (2021b); this chapter might be of interest as the authors also depict the nematode fossil record through the Phanerozoic and clearly demonstrate that amber provides one of the richest records of nematode body fossils including parasitic ones.

Thanks. We added this reference here.

Line 76-79: Indeed, also the changes in proportion for heterometabolous to holometabolous insects could be mentioned. I feel that you undersell the importance of your study by dumping this information in the supplementary material. You also allude to a statistical analysis in the abstract but as far as I can see you are just naming percentages and have pie diagrams which can be widely deceiving (e.g., Rougier et al. 2014). Depicting the changing proportions of parasitic nematodes from heterometabolous to holometabolous insects would make your study easier to follow and reproduce as well as your interpretations more convincing.

Thanks. We have added a new Figure 9B and Table 1 to our paper.

Lines 100, 114, 128, 143, 157, 174, 192, 210, 224, Below each new species, the zoobank number should be added.

Thanks. Added.

Line 300: You need to back up this statement with a reference. De Baets et al. 2021 (not to be confused with the other reference you already cite) recently depicted the known record of nematode body fossils and show that amber is one of its richest sources of nematodes backing up your statement.

Thanks. This reference is informative and we added this reference.

Line 302-305: This is an important result and therefore the information should not be buried in the supplementary material. It is crucial to analyze/depict this information in a bar plot rather than pie diagrams which although graphically appealing might distort information (compare Figure 1.3 in De Baets et al. 2021) and I feel this could be done by separating those species in heterometabolous and holometabolous insects per amber deposit. To make your interpretations more robust and deal with differences in sampling heterometabolous versus holometabolous insects, at minimum binomial error or confidence intervals should be added to this plot (Raup 1991; Takeda and Tanabe 2014) if not more broadsweeping statistical analyses. Confidence intervals are more conservative and can easily be calculated in Matlab with Binofit function or R with the binom.confint function of the package Binom as well as other software. I feel such analyses could be done for both mermithids and all (parasitic) nematodes in those 3 amber deposits. Particularly tabulated data and depiction of the distribution of mermithids in modern insects would be helpful to understand the context. This might be trivial to you but not so to the reader who does not necessarily has access to all relevant literature.

Thank you very much for pointing out these issues. We also realize this drawback and we have now calculated the 95% CI using the Agresti-Coull method of the “binom.confint” function from the binom R package (https://cran.r-project.org/package=binom) of R 4.2.2. We also added a new Figure 9B and Table 1 in our paper to address this problem. We did not prepare a similar figure for mermithids alone because the dataset is too small at present. Meanwhile, since we compiled the “occurrence” of invertebrate–nematode associations from these amber localities, it is impossible to compare with modern mermithids. For example, the parasite of Cretacimermis chironomae occurs five times in Kachin amber, but an extant dipteran-parasitized mermithid species can occur many times just in a single pond. However, it is evident that mermithids as well as all invertebrate-parasitized nematodes prefer to infect holometabolous insects rather than other invertebrates (Poinar, 1975; Poinar, personal observation). We have also added some explanations in our paper, please see lines 367–369 in the clean copy.

Line 330-331: In addition to De Baets et al. 2021a, 2021b would be highly relevant in this context.

Thanks. We have added this reference here.

Line 344-353: I appreciate this explicit ethics statement and I feel it should be mandatory in all kind of studies dealing with fossils not just amber.

Thanks for your positive comment.

Line 390: Thank you for providing the raw data but I feel your study would also benefit from depicting this data in a bar plot with confidence intervals (as explained before) as well as add data and host distributions of modern mermithids.

Thanks. We have added a new Figure 9B and Table 1 to our paper.

Author details

1. Cihang Luo

   1. State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology and Center for Excellence in Life and Paleoenvironment, Chinese Academy of Sciences, Nanjing, China
   2. University of Chinese Academy of Sciences, Beijing, China

   Contribution

   Conceptualization, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   chluo@nigpas.ac.cn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4855-6185

2. George O Poinar

   Department of Integrative Biology, Oregon State University, Corvallis, United States

   Contribution

   Investigation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

3. Chunpeng Xu

   1. State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology and Center for Excellence in Life and Paleoenvironment, Chinese Academy of Sciences, Nanjing, China
   2. University of Chinese Academy of Sciences, Beijing, China

   Contribution

   Investigation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

4. De Zhuo

   Beijing Xiachong Amber Museum, Beijing, China

   Contribution

   Investigation, Visualization

   Competing interests

   No competing interests declared

5. Edmund A Jarzembowski

   State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology and Center for Excellence in Life and Paleoenvironment, Chinese Academy of Sciences, Nanjing, China

   Contribution

   Writing – review and editing

   Competing interests

   No competing interests declared

6. Bo Wang

   State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology and Center for Excellence in Life and Paleoenvironment, Chinese Academy of Sciences, Nanjing, China

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   bowang@nigpas.ac.cn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8001-9937

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