Kikiki huna Huber & Beardsley

Huber, John T. & Noyes, John S., 2013, A new genus and species of fairyfly, Tinkerbella nana (Hymenoptera, Mymaridae), with comments on its sister genus Kikiki, and discussion on small size limits in arthropods, Journal of Hymenoptera Research 32, pp. 17-44 : 24-31

Kikiki huna Huber & Beardsley Figs 2 23 48

Description.

Female. Body length (critical point dried specimens) 158-190 μm (n=10). Antenna. Funicle segments and basal claval segment without mps, apical claval segment with 3 mps ( Figs 24 , 43 ). Antennal length/width measurements (n=4, Costa Rica specimens): scape, 36-47/10-11, pedicel 19-25/12-13, fl1 8-9/5-6, fl2 16-18/6, fl3 14-17/6-7, fl4 12-18/7-8, clava 46-54/15. Wings. Fore wing ( Fig. 27 ) length 182-226, width 20-24, length/width 9.10-9.24, longest marginal setae 102-123 (n=3, slide mounts), hind wing ( Figs 23 , 27 ) length 162-198, width 4-5, longest marginal setae 96 (n=1-3).

Male. Unknown for Neotropical region.

Material examined.

20♀. COSTA RICA. Heredia. La Selva Biological Station, 10°26'N, 84°01'W, 75m, 27-28.ii.2003, J.S. Noyes (1♀, CNC). Puntarenas. La Gamba Biological Station, 8°42'N, 83°12'W, 150m, 13-14.ii.2006, J.S. Noyes (1♀, BMNH); Reserva Absoluta Cabo Blanco, 9°35'N, 85°36'W, 30m, 16-17.ii.2009, J.S. Noyes, sweeping (9♀, CNC, INBio, UCRC); Reserva Privada Karen Morgensen, 9°52'N, 85°03'W, 305m, 23-24.ii.2007, J.S. Noyes, sweeping (6♀, BMNH). HAWAIIAN ISLANDS. Molokai I.: Mapulehu (1♀ paratype, CNC). TRINIDAD & TOBAGO. Trinidad. Curepe, Santa Margarita Circular Road, 8.xii.1974-2.ii.1975, F.D. Bennett (2♀, CNC).

Discussion.

We cannot find any morphological differences suggesting that the specimens from Costa Rica and Trinidad are different from the Hawaiian specimens. The number and distribution of mps on the antennal segments as reported by Huber and Beardsley (2000b) are incorrect, as mentioned above. Both the Hawaiian and the Neotropical American specimens have the same mps distribution. The body length of the former averages slightly larger, from 190-ca 330 μm long ( Huber and Beardsley 2000b) but this is insufficient evidence for species separation.

At the genus level, and even the species level, the Hawaiian fauna at low elevation appears to be almost entirely represented by exotic species ( Huber and Beardsley 2000a, Beardsley and Huber 2000, Triapitsyn and Beardsley 2000), except for one genus ( Polynema ) that has numerous, native species at higher elevations. Although Kikiki huna appeared to be endemic( Huber and Beardsley 2000b), this is simply because specimens of the genus had not yet been collected elsewhere-not surprising given their minute size. Specimens have since been found in Argentina ( Luft Albarracin et al. 2009), Australia ( Lin et al. 2007) and Costa Rica (this paper) indicating that the genus is widespread. Given its mostly low elevation range in the Hawaiian Islands it was almost certainly accidentally introduced from elsewhere. We therefore treat all the specimens as the same species and suggest Kikiki huna in the Hawaiian Islands came originally from Central America.

Tinkerbella would key to Kikiki in Huber (2009b) because at the time he thought that the genus included species with a variable number of tarsomeres (3 or 4) and, in females, funicle segments (4 or 5) and clava segments (1 or 2). Because Tinkerbella is distinct on other features as well it is described here as a new genus. Huber (2009b) and Lin et al. (2007) included Kikiki in the Alaptus group of genera, mainly because of its minute size. If the key in Huber (2009b) is used, couplet 1 should be deleted and replaced with the following two couplets at the beginning of the key to separate Tinkerbella and Kikiki from the remaining Alaptus -group genera, and to distinguish them from each other.

1	Venation long, clearly more than half wing length; tarsi 3- or 4-segmented	1a
-	Venation short, clearly less than half wing length; tarsi 5-segmented	couplet 2 in Huber (2009b)
1a	Clava 2-segmented, funicle 4-segmented; tarsi 3-segmented	Kikiki Huber and Beardsley
-	Clava entire; funicle 5-segmented, tarsi 4-segmented	Tinkerbella Huber and Noyes

However, several features of both genera show that their placement in the Alaptus group of genera is wrong and that they are best placed in the Anagrus -group because they share at least seven features with some or all of those genera: frenum apparently longitudinally divided by a groove, petiole and base of gaster distinctly narrower than propodeum and middle of gaster, tarsi with at most 4 tarsomeres (3 in Kikiki ), stemmaticum present, mandibles with 4 teeth, fore wing venation with proximal macrochaeta much shorter than distal macrochaeta, and second phragma not projecting past posterior margin of propodeum.

Both Kikiki and Tinkerbella are distinguished from other Anagrus -group genera by the venation clearly longer than half the wing length and hind wing essentially without fringe setae on the anterior margin. The other genera in the group have the venation clearly less than half wing length and the hind wing fringed with fairly long setae on the anterior margin. No member of the Anagrus group of genera is anywhere near as small as Tinkerbella or Kikiki so these genera can be distinguished on body size alone.

Some members of the Alaptus or Camptoptera groups of genera are also extremely small but except for Alaptus borinquensis none has been found that equal minute size of Tinkerbella or Kikiki , i.e., less that about 250 μm. One specimen of Alaptus from the same collecting event in Costa Rica as Kikiki huna measured 272 μm and species of Eofoersteria (also with 4-segmented tarsi, despite being in the Camptoptera group) are about 320 μm. The six specimens (4 females, 2 males, on 4 slides, USNM) of Alaptus borinquensis are uncleared and mounted mostly in lateral view in Canada balsam, and some are slightly shrivelled. The smallest specimen, a male, measured 203 μm in length, not 186 μm as reported by Dozier (1932). The ranges for females and males, respectively, are 215-411 μm (n=4) and 203-311 μm (n=2), measured with a filar micrometer at 400 × by JTH.

Body size limits in arthropods

What is the smallest size that an adult insect can attain, as measured by body length, and/or fore wing length for flying insects? If something is physically possible in living things some individuals of at least one species, extinct or extant, will likely have achieved it. So the lower size limit, by whatever measure of size is chosen, was almost certainly already evolved-somewhere, sometime. If we have not already found them, we must surely be close to discovering the smallest insects and other arthropods.

The minimum size possible for invertebrates with articulated appendages of locomotion (superphylum Arthropoda ) that would allow crawling, walking, or active flight (wing flapping, not gliding) is determined by two types of constraint: internal (physiological and structural) and external (physical). For multicellular animals, the size and structure of cells cannot go below a certain minimum before they cannot function for the purpose intended. Therefore, once any type of cell has attained its minimum size, the number of cells making up a particular tissue or organ must decrease as the animal becomes smaller. But there evidently is a lower limit to cell number in any particular organ for it to function as intended, perhaps as low as one, e.g., a single muscle fiber or ommatidium. Or the tissue or organ may be dispensed with entirely, usually because it is no longer necessary, e.g., eye loss in obligate cave-dwelling species (troglobites). Once these internal constraints are reached the arthropod cannot become any smaller. Nuzzaci and Alberti (1996) showed that Eriophyidae (Acari) have no respiratory system and no striated muscle [in contrast to insects and other Acari, which have striated muscle exclusively ( Beinbrech 1998, Alberti and Coons 1999)]. Polilov (2007) discussed miniaturization related features in Mymaridae , and Polilov (2012) showed that neurons of Megaphragma mymaripenne Timberlake are anucleate. Fischer et al. (2011) discussed size limits in ommatidia in a small parasitic wasp, Trichogramma evanescens Westwood. Grebennikov (2008) reviewed the limiting factors for small size in arthropods.

As body size decreases, external constraints, e.g., desiccation ( Neville 1998), surface tension, and fluid viscosity become relatively more important. Thus, in minute organisms the muscle strength needed to power an articulated appendage for active locomotion is determined not only by internal factors such as the minimum muscle cell size and cell number but also by external factors such as viscosity of the fluid (air or water) in which active movement occurs. Although surface area to volume ratio increases with decreasing size allowing small organisms to be relatively stronger than larger organisms, there still comes a point at which muscles are so small that they cannot power an articulated appendage in a medium that, for their size, must be quite viscous. Yet, external physical factors such as air viscosity are likely still not a constraining factor because even the smallest walking or flying insects appear to be large enough to overcome them. Their problem is to overcome their own inertia, not viscosity of the fluid in which they move. For even smaller organisms than arthropods, viscosity and surface tension may finally become the limiting factor. These organisms do not (and cannot) have articulated appendages of locomotion, particularly if the appendages have intrinsic muscles that move the various segments independently.

Even with the increased mechanical efficiency resulting from smaller body size and energy conservation efficiencies conferred by Weis-Fogh clap-and-fling flapping ( Weis-Fogh 1973, Miller and Peskin 2005), the elastomeric protein resilin ( Neville 1998, Elvin et al. 2005), the natural elasticity of muscle and cuticle itself, and light cuticular wings ( Neville 1998) it is difficult to believe for winged insects that such a small size as occurs in Kikiki huna is possible. The fact that some specimens of Megaphragma are about the same minimum size as some specimens of Kikiki suggests that winged insects have indeed reached their limit for size reduction.

A diversity of other very small insects capable of active flight have the wing surface (membrane) reduced and wing fringes, especially of the hind wing, greatly lengthened, e.g., species of Thysanoptera , various families of small Lepidoptera and parasitic Hymenoptera , and Ptiliidae ( Coleoptera ). While this may slightly reduce wing weight the reduced wing surface and relatively long setae are more likely to have an aerodynamic function, perhaps to reduce turbulence and hence drag on a wing flapping at several hundred beats per second. Interestingly, in Kikiki ( Fig. 27 ), Tinkerbella ( Fig. 6 ) and Megaphragma ( Fig. 51 ) the fringe setae along the leading edge of the hind wing are absent or almost so, whereas in other genera of Trichogrammatidae and Mymaridae they are present, albeit short. The separation of muscles that power flapping, i.e., the indirect flight muscles that cause thoracic distortion, resulting in wing flapping, from the direct flight muscles that control wing pitch may also be important in allowing insects capable of active flight to attain a minute body size.

Relatively long legs powered by sufficiently strong muscles to elevate the body may be linked to and necessary for active flight. The muscles required to move articulated legs to enable walking by lifting the entire body off the ground and moving forward must have a lower size limit or the legs could not be used for that purpose. Two sets of opposing intrinsic muscles are needed: extensor/elevator muscles strong enough to lift the entire body sufficiently above the substrate to initiate flight, perhaps by allowing a wing stroke of more than 90°, and flexor/depressor muscles to allow walking, and perhaps secondarily to draw the legs against the body during flight to reduce drag. High speed photography of Trichogramma lifting off a substrate ( Lentink and Voesenek 2012) suggests that the first wing stroke is greater than 90° and, once airborne, that individuals in flight move forward by pushing air forcefully backwards, especially on the up stroke. With about 350 wing beats per second Trichogramma individuals keep themselves in a reasonably high Reynolds number range. But the video clip does not seem to show that the legs are used to leap vertically off the substrate prior to initiating a wing flap. In these wasps the legs need only be long and strong enough to lift the entire body off the ground for normal walking and perhaps to allow the first wing flap to be greater than 90°, sufficient to allow lift off. Trichogramma spp, have relatively wide wings, (200-300 μm), with a fore wing width to length ratio about 0.5 and wing fringes that are about 0.2 × wing width. These are quite unlike Kikiki , with a forewing width of 20-25 μm, a width to length ratio of 0.11 and fringes about 5.0 × the wing width, so flight aerodynamics between the two genera may be very different.

In insects with free living adults and larvae the lower body length limit seems to be about 400 μm ( Grebennikov 2008), imposed by the need to have a sufficiently large egg to nourish the developing larva sufficiently so it can be free-living, i.e., it must have the necessary initial body resources to move around actively upon hatching, perhaps to search for food and avoid potential predators. Females of the smallest oribatid mites (Acari) also lay only a single, relatively enormous egg at a time, e.g., a species of Brachychthonius ( Brachychthoniidae )with a female 180 μm long (distorted by compression of the coverslip) has an egg of 100 μm (D. Walter, pers. comm.). The smallest known fly is also 0.4 mm long ( Brown 2012).

The constraint of minimum egg size as a determinant of minimum body size does not apply to parasitic insects. Eggs in these can be much smaller because the larvae hatch inside the host. They do not have to search actively for food because it completely envelops them. The limiting factor to small size in adult parasitic insects must therefore be minimum cell size and sufficient cells of each type of tissue, as discussed above. Females of Tinkerbella , Kikiki , and Alaptus borinquensis are the smallest Mymaridae , and some specimens of Kikiki are the smallest recorded winged insects. The body length of five specimens from Costa Rica is 158 μm (1 female), 160 μm (2 females), and 170 μm (2 females). Three specimens are thus smaller than the smallest recorded females of Megaphragma caribea , the previous record holder at 170 μm ( Delvare 1993).

The next step in body length reduction in insects is in Dicopomorpha echmepterygis . Females are winged and relatively long (one measured 386 μm dry but not shivelled and 550 μm on a slide mount), but the males slide-mounted males were 139-240 μm long (n=8) ( Mockford 1997). One critical point dried female (CNC) measured by JTH is 340 μm long and two critical point dried males (BMNH, CNC) from the remaining 10 male paratypes, measured independently by JTH and JSN are 126.17 μm and 130 μm, respectively (ESEM measurement). Males have significant morphological reductions or losses. They lack eyes and ocelli, and the appendages are greatly reduced (antenna, tarsi) or absent (wings, mouthparts) ( Figs 52-55 ). Because they cannot feed their only energy source is what has been stored as larvae, which would have obtained all their nutrients from their psocopteran host egg. The leg segments of males are strangely disproportionate, with huge coxae ( Figs 52 , 53, 55) as long as the femora, protibiae shorter than the femora, and the tarsi absent except for the large, bell-shaped arolium on each ( Figs 52, 55 ) that presumably acts as suction cups to attach to females. Because the legs, especially the hind legs, are long relative to the body length, males clearly can raise their entire body above the substrate in order to walk more or less normally over the short distance necessary to find a female. The tarsal structure, and the fact that some males were found attached to females ( Mockford 1997), show that males are phoretic and need only walk only far enough to crawl onto a female to copulate, almost certainly while the female is still in the egg (females presumably emerge from the same host egg or egg cluster so the distance traversed by a male is very short). Males evidently have enough energy to do this and probably nothing else.

Further reductions in body length occur in terrestrial Arthropoda other than insects. In mites, the smallest adult individuals of several species in three families are less than 95 μm in length: Cochlodispus minimus Mahunka at 79 μm ( Mahunka 1976) and Microdispus australis Mahunka at 82 μm ( Mahunka 1969) (both Microdispidae ), female of Indosetacus rhinacanthi Ghosh and Chakrabarti at 86 μm ( Ghosh and Chakrabarti 1987), male of Eriophyes parvulus (Nalepa) at 90 μm ( Nalepa 1892), both sexes of Achaetocoptes quercifolii Farkas at 90 μm ( Farkas 1961) (all Eriophyidae ), and females of Iponemus truncatus eurus Lindquist at 93 μm and Iponemus confusus oriens (Lindquist and Bedard) at 94 μm (both Tarsonemidae ) ( Lindquist 1969). That small size in arthropods is not a recent evolutionary phenomenon is shown by a Triassic mite, Ampezzoa triassica Lindquist and Grimaldi ( Eriophyidae ), 124 μm in length ( Schmidt et al. 2012). Adults of Eriophyidae have only two pairs of usually 5-segmented legs yet they are evidently capable of locomotion but the legs are so reduced that they cannot lift the entire body off the substrate. “Walking” in Eriophyidae is an inching or looping motion. The more or less worm-like body is arched between the two pairs of forelegs anteriorly and the terminal sucker posteriorly, and alternate gripping and releasing by the legs and sucker allow the mite to inch along. True walking using the legs only presumably does not occur and they probably do not move much by this method during their life time. Instead, dispersal is by aerial drifting. However, Microdispidae and Tarsonemidae have 4 pairs of legs and are capable of normal walking. The smallest adult of Neoliochthonius piluliferus (Forsslund) ( Brachychthoniidae ) is 123 μm ( Forsslund 1942). If a mite doubles in size with each moult or increases in body length by a factor of about 1.3 ( Hutchinson’s Ratio-applied to comparison of different life stages within a species instead of comparing competing species in the same habitat) a larva or protonymph would be as small as about 50 μm in length. A larva of this length was found by D. Walter (pers. comm.). Whereas Insecta have two sets of opposing intrinsic muscles in their leg segments ( Fig. 47 ) as indicated above, Acari only have flexors (except for those moving the apotele, at the apex of the tarsus). In all Acari the distal segments flex due to muscular contraction and extend due to hydrostatic pressure ( Alberti and Coons 1999).

For comparison with terrestrial arthropods, larvae of the marine parasites of Copepoda Stygotantulus stocki Boxhall and Huys at 94 μm ( Boxhall and Huys 1989) and Tantulus dieteri Mohrbeck, Arbizu and Glatzel ( Crustacea : Tantulocarida , Basipodellidae ) ( Mohrbeck et al. 2010, Martin and Davis 2001) at ca. 85 μm are the shortest. Notably, all members of the subclass lack recognizable cephalic limbs, other than paired antennules in one known stage ( Martin and Davis 2001). The loss of appendages and the parasitic life style of adults means that the much higher viscosity of water compared to air is irrelevant in impeding locomotion, because the immature stages evidently disperse by passive drifting (as do Eriophyidae ), and adults are parasitic so evidently do not move.

Below a certain body length it is useless to have articulated appendages because the segments could not be moved relative to one another, or the entire appendage relative to the body, by intrinsic muscle power alone. Instead, if appendages of locomotion exist at all (e.g., pseudopods), they would be short and wide, would not be articulated, and would be moved instead by body muscles causing hydrostatic changes in pressure, combined perhaps with flexor muscles originating within the body but attached near or at the appendage apex. The length of the larva of Neoliochthonius piluliferus rivals some Rotifera, also as short as 50 μm, suggesting that at about this size the changeover from locomotion by partial muscle power intrinsic to leg segments (Acari) to hydrostatic power alone (Rotifera, other non- Arthropoda ) may occur.

We suggest that the smallest winged insects capable of flapping flight could not be less than about150 μm in length, and the smallest capable of normal walking (body lifted entirely of substrate) not below about 125 μm. Among insects, Kikiki huna may well have attained the lower limit for active flight and Dicopomorpha echmepterygis the lower limit for normal walking. Among other arthropods capable of walking, 80 μm is suggested as the lower limit for adults and ca. 50 μm for immatures.

Figures

Figures 1, 2. Habitus, lateral. 1 Tinkerbella nana female 2 Kikiki huna, female. Scale line = 100 μm.

Figures 23 - 26. Kikiki huna female, on slide (cleared, except Fig. 23). 23 habitus, dorsal 24 head + right antenna, anterior 25 head, posterior 26 mesosoma, dorsal + metasoma, dorsal but focus at lower plane to show ovipositor. Scale line = 100 μm.

Figures 43 - 48. Kikiki huna, female, micrographs except Fig. 47. 43 antenna, lateral 44 mesosoma, dorsal 45 frenum - anterior half of metasoma, dorsal 46 metasoma, dorsal 47 hind leg, uncleared lateral, showing muscles; 48, right metatarsus, dorsal. Scale line = 20 μm, except Fig. 47 = 50 μm and 48 = 10 μm.

Figures 27 - 29. Kikiki huna female, cleared on slide. 27 wings 28 metasoma, dorsal surface 29 metasoma, ventral surface (seen dorsally through cleared metasoma). Scale line = 100 μm.

Figures 5 - 7. Tinkerbella nana female paratype, cleared on slide. 5 head + right antenna, anterior 6 wings 7 mesosoma + metasoma dorsal, and ovipositor ventral (inset). Scale line = 100 μm.

Figures 49 - 51. Megaphragma sp., uncleared on slide. 49 head + antenna, anterior (note black eyes) 50 mesosoma + metasoma, dorsal (note huge mesophragma, hence the genus name) 51 wings + middle leg (note 3 - segmented tarsi, diagnostic for Trichogrammatidae). Scale line = 100 μm.

Figures 52 - 55. Dicopomorpha echmepterygis male, paratype, ventral. 52 habitus 53 head + prothorax + procoxa 54 apex of gaster 55 mesosoma + base of most legs and metasoma. Scale line = 20 μm, except Fig. 52 = 50 μm.