Mechanisms of ovipositor insertion and steering of a parasitic wasp

Urosˇ Cerkvenika,1, Bram van de Straata, Sander W. S. Gusseklooa, and Johan L. van Leeuwena

aExperimental Zoology Group, Wageningen University and Research, 6708WD Wageningen, The Netherlands

Edited by Raghavendra Gadagkar, Indian Institute of Science, Bangalore, India, and approved July 28, 2017 (received for review April 13, 2017) Drilling into solid substrates with slender beam-like structures Buckling is a mechanical failure of a structure which occurs, for is a mechanical challenge, but is regularly done by female para- instance, when a beam cannot withstand the applied axial load sitic wasps. The wasp inserts her ovipositor into solid substrates and bends, possibly beyond its breaking point. As buckling occurs to deposit eggs in hosts, and even seems capable of steering more easily in slender beams, this is a real danger for parasitic the ovipositor while drilling. The ovipositor generally consists of wasps. Buckling depends on four parameters: (i) the axial load three longitudinally connected valves that can slide along each applied on the beam, (ii) the second moment of area of the beam, other. Alternative valve movements have been hypothesized to (iii) how well is the beam fixed on both ends (i.e., “free to slide be involved in ovipositor damage avoidance and steering during sideways,” “hinged,” or “fixed”), and (iv) the length of the beam. drilling. However, none of the hypotheses have been tested in During puncturing, axial loading of the ovipositor cannot be vivo. We used 3D and 2D motion analysis to quantify the probing avoided, so only the other factors can be adjusted. The sec- behavior of the fruit-fly parasitoid Diachasmimorpha longicau- ond moment of area is largely determined by the diameter of data (Braconidae) at the levels of the ovipositor and its individual the ovipositor and its wall thickness. To simplify insertion, the valves. We show that the wasps can steer and curve their oviposi- ovipositor must be as thin as possible, while the internal channel tors in any direction relative to their body axis. In a soft substrate, needs to be big enough for an egg to pass. Both of these require- the ovipositors can be inserted without reciprocal motion of the ments increase the chance of buckling. In all wasps, the oviposi- valves. In a stiff substrate, such motions were always observed. tor is fixed internally to the reproductive system and the muscles This is in agreement with the damage avoidance hypothesis of that move the ovipositor (4, 19), so very little variance can be insertion, as they presumably limit the overall net pushing force. expected related to the fixation of the ovipositor. A parameter Steering can be achieved by varying the asymmetry of the distal that can be changed is the “functional” length of the oviposi- part of the ovipositor by protracting one valve set with respect to tor. Some wasps protrude only a small part of the total ovipos- the other. Tip asymmetry is enhanced by curving of ventral ele- itor outside their bodies before puncturing the substrate. The ments in the absence of an opposing force, possibly due to pre- part retained in the abdomen is then either strongly coiled or tension. Our findings deepen the knowledge of the functioning telescopically retracted (20, 21). In other species, the functional and evolution of the ovipositor in hymenopterans and may help length of the ovipositor is reduced by supporting it by clamping to improve man-made steerable probes. the ovipositor with parts of their hind legs (11, 18, 22) or with specialized sheaths (1, 10, 23, 24). Diachasmimorpha longicaudata | ovipositor kinematics | buckling Little is known about the mechanisms parasitic wasps use for avoidance | spatial probing | minimally invasive probe further insertion and buckling prevention of the ovipositor after the initial puncturing of the substrate. Vincent and King (23) hypothesized a mechanism that wasps might use based on the rom a mechanical perspective, it is very difficult to drill into Fa solid substrate with a very thin probe, because it can easily bend and break. Parasitic wasps, however, do this regularly when Significance they use their slender ovipositors to search for hosts in solid sub- strates, such as fruits or even wood (1–3). Using slender probes to drill through solids is challenging, but The general morphology of the ovipositor is similar across all desirable, due to minimal disturbances of the substrate. Par- wasp species (4, 5); it consists of four elements, called valves, asitic wasps drill into solid substrates and lay eggs in hosts of which two are often merged such that three functional valves hidden within using slender probes and are therefore a good remain (Fig. 1). In most species, the distal part of the ovipositor model for studying mechanical challenges associated with this is morphologically distinct (3, 6), which we will refer to as the process. We show that wasps are able to probe in any direc- tip. The valves can slide along each other (5, 7) and do not get tion with respect to their body orientation and use two meth- dislocated under natural conditions, because they are longitudi- ods of insertion. One of the methods implies a minimal net nally connected via a tongue-and-groove mechanism (5, 8–10). pushing force during drilling. Steering was achieved by adjust- The ovipositor and the “wasp waist,” a constriction of the body ing the asymmetry of the probe’s distal end. Knowledge on between the first and second abdominal segment (11), are essen- probing mechanisms of wasps is important for the under- tial in probing behavior and are therefore considered to be standing of the hymenopteran evolution and for the devel- instrumental in the evolution of the order (11–15). The shape, opment of minimally invasive steerable probes. structure, and mechanical properties of the ovipositors are puta- tively adapted to the substrates into which the animals need Author contributions: U.C., S.W.S.G., and J.L.v.L. designed research; U.C. and B.v.d.S. per- to probe (6, 16–18), and because both substrates and hosts are formed experiments; U.C., B.v.d.S., S.W.S.G., and J.L.v.L. analyzed data; and U.C., S.W.S.G., so diverse, this might have resulted in high species diversifica- and J.L.v.L. wrote the paper. tion of the hymenopterans (13, 14). However, to understand the The authors declare no conflict of interest. observed diversity in the ovipositor shapes, understanding of the This article is a PNAS Direct Submission. probing mechanics is essential. Data deposition: The data reported in this paper have been deposited in Dryad Wasps are faced with two problems when searching for hosts in (doi.org/10.5061/dryad.8bc95). (solid) substrates: (i) how to insert the ovipositor without buck- 1To whom correspondence should be addressed. Email: [email protected]. ling/breaking it and (ii) how to maneuver with the ovipositor to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. reach the target. 1073/pnas.1706162114/-/DCSupplemental.

E7822–E7831 | PNAS | Published online August 28, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1706162114 Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 fteeeet 3,3) h lmnspsesacranlvlof level certain a possess elements The 36). (35, elements bending (pre)tension the that from assumed of originate is mouthparts It hemipteran other. in each along moments slide to able longitudinally are interconnected and of are consist that elements mouthparts slender hemipteran multiple bending the ovipositors, active the mouthparts. of hemipteran to of Similar mechanism control the possible for hypothesized third bend- been has to The leads 2D). which (Fig. nodes, valves with dorsal ing align or arches ventral the the protracted, ovipos- When are and the straight. and arches aligned approximately the are is valves rest, itor ventral At and dorsal nodes. the flexible of nodes and sclerotized alternated less bending arches, with this stiff sclerotized, on heavily relying of ovipositors consists of mechanism part scleroti- distal valve The in (34). differences zation on second relies A 2C). mechanism (Fig. bending valves active to the individual due in occurs limit compression Bending and 33). structures tension (32, valves anatomical individual special of range mechanism, motion first the In (31). needles steerable proposed of been generation may has new bevel what a to adjustable for similar An bending, of valves. degree chang- the the as by of control enhanced function positions be relative potentially presumably species the thus can across ing shape can bevel ovipositors and 27). The most 30) bevel. (26, a 29, of path dur- (6, tips straight direction asymmetric tip a The are the from adjust (28). to away insertion used be tip ing can the bevel the push acting of tip Rotation forces a asymmetric The such 2B). on (Fig. tip beveled asymmetric valves. ovipositor the originate of moments movements bending relative sub- the when from the occurs with bending ovipositor Active inserted strate. the of during interactions mechanical direction 2 pas- (Fig. steer- into ones tip divided active a tar- and be sive and the can desired mechanisms flexibility bending the adjusts needs Proposed of probing. ovipositor that direction the mechanism so, the ing/bending do in To tip (25). ovipositor get the steer to (23). ovipositor the loads damage axial could and the forces that push into net inserted excessive further avoiding is while the ovipositor substrate, the limiting valves, by the and protraction of avoided the retraction alternating hypoth- is By the valve motion. forward to protracted for of amplitudes According the guides inward. of as pushed buckling serve is esis, valves that valves, These valve three third anchors. on the the as structures of hook-like function the two tips of on their because force stationary mech- kept pulling proposed are the a which In apply 2A). wasps (Fig. anism, ovipositor the of morphology (B, scan. micro-CT 100 bar: a [Scale with valves. 10 three obtained and the (A) ovipositor showing ovipositor the the of of Cross-section part a of tion in shown Region view. 1. Fig. ekei tal. et Cerkvenik A dorsal he ehnsshv enpooe o ciebending. active for proposed been have mechanisms Three an has ovipositor an when occurs presumably bending Passive need wasps the that is oviposition during challenge second The vpstrof Ovipositor (B, µm ventral Inset E mg fteoioio;side ovipositor; the of image SEM (A) longicaudata. D. B )]. Dreconstruc- 3D A (B) lines. dashed with indicated is B .Psiebnigoiiae from originates bending Passive B–E). ventral dorsal Inset µm ) n teigaeue yprstcwss ed hsuigthe using this do We insertion wasps. of (range, parasitic methods by use proposed species used ovipositor the are of steering the density which and substrate quantify to determine relation to to in and probing) aim one of substrate. curvature no we the and but speed, inside work, 37), probing this of (25, dynamics substrate In the the focus- analyzed studies inside probing ever few ovipositor has of a the only theories with on proposed data, ing of morphological The on properties based 16–18). mechanical are (6, num- quantifying small ovipositors studies a the empirical only is of there ber hymenopterans, of success lutionary curvature the control insertion. to during way ovipositor a the offers of with and bending correlates of probably amount valves the the the of of other amplitude retraction the the indi- and mechanisms, protraction toward an three all inward When In curves 2E). structure. (Fig. tip elements straight its counter- a protracts, they in element opposed. so vidual resulting tips not their other, when with side each aligned one act are to elements the curve rest, to At tend and tension inner he ap i o rb nbt usrts eaaye only analyzed We inserted substrates. wasps both where in instances probe not did see wasps parameters Three gel of stiff- calculations and and 1 on densities Table in gel presented different (parameters two nesses with wasps 28 presented We Results steerable novel of development (38–43). the tools man-made in surgical of help development also knowledge presumably the Such probing. will in or probes. insertion, applied tunneling, slender be for the instruments with can on turn, drilling acting in for add demands This, mechanism will functional the study the our and of addition, ovipositor simi- In understanding use food. the they for to as probe mosquitoes, to and structures lar and hemipterans probing hymenopterans of parasitic into possibly of insight and groups provide other also of Extrap- possibilities will ovipositor. steering results slender our and of long its olation of because example lent iee elgbe rcinaogtesat(F shaft (F the friction along Inner clarity). Friction for negligible. shown are sidered valves two ( (only forces. (23) pull anisms arrows empty and forces push represent 2. Fig. fidfo e.35). (mod- ref. mouthparts hemipteran from in ified observed as text protraction see their (34); upon curving segments valve (E oviposi- 34). of Arched ref. sclerotization from (D) (modified differential 32). to ref. due to from bend (modified leads tors forces needle compres- arrows) and a black arrows) (small (gray of tensile sion to shape due push- bevel bending causes the A (32) (F displacements of (B) forces tip forces 23). asymmetrical to (cutting) ref. due bending hook from and (modified friction valve ing the counteract and strate (F forces hook the with together ept h rpsdiprac foioiosfrteevo- the for ovipositors of importance proposed the Despite ULPUSH PULL F F A h f yohssdisrinadsern ehnssi D ularrows Full 2D. in mechanisms steering and insertion Hypothesised hc rvdsa excel- an provides which longicaudata , Diachasmimorpha F in F B f PNAS rtnino niiuleeet 3)last in- to leads (35) elements individual of Pretension ) F tip | h ,ke h vpstracoe ntesub- the in anchored ovipositor the keep ), ulse nieAgs 8 2017 28, August online Published D CE 0 fteroioio inside ovipositor their of >60% tip .(C ). IMtrasadMethods. and Materials SI f etito ninter-element in Restriction ) ftetopligvalves, pulling two the of ) h uhpl mech- push–pull The A) .Frdetails For S1). Fig. in | scon- is ) E7823

PHYSIOLOGY PNAS PLUS Table 1. Gel parameters. Storage (G0), loss (G00), dynamic shear curved insertions. The retractions were sometimes also used to (G∗), and elastic (E∗) moduli make minor adjustments to the direction of insertion. Conc. G’, kPa G”, kPa G*, kPa E*, kPa Probing Range. We calculated the maximum arc length of each 2% 11.970 ± 0.734 0.676 ± 0.278 11.989 35.967 insertion trajectory, as well as the radius (r), depth (d), and posi- 4% 68.544 ± 2.016 4.615 ± 1.132 68.700 206.099 tion vector (R) of the respective endpoints. We took the trajec- ◦ Conc., concentration. tory insertion angle α (deviation from 90 ) as the angle between the R and the vertical vector along the depth axis (Fig. 3B). From a single horizontal body orientation, wasps were able to the substrate. For three of the animals, the top camera record- probe in all directions within the gel (Fig. 3C), and we observed ing their orientation during probing stopped working. Their data no directional preference of insertions belonging to the same ses- were excluded from the calculations of the range of probing, but sion (Fig. S2). No clear directional preference was seen in the were included in the velocity analysis. This amounted to 107 and combined behavior of all animals (Rayleigh test, P = 0.5 and 92 insertions used for range calculations, and 117 and 113 inser- P = 0.05 for 2% and 4% gels, respectively). tions for speed calculations in 2% and 4% gels, respectively. The correlation between the vertical and horizontal com- ponent of the position vector (Pearson’s correlation = 0.57, Description of Probing Process. When starting to probe, an individ- P < 0.001) shows that a deep probe had a large chance of having ual wasp lifted its abdomen, oriented the still-sheathed oviposi- a limited horizontal amplitude and vice versa (Fig. 3D). tor vertically, and punctured the substrate with the most distal In general, the probing space of the wasps can be visual- part of the ovipositor tip. While inserting the ovipositor deeper, ized as a cone with a curved base. The radius of the cone is the sheaths peeled away from the ovipositor base into a hairpin- substrate-dependent and is larger in the 2% gel than in the 4% like structure (Fig. 3A). Often, the wasp partially retracted the gel (respective medians: 1.90 mm and 1.19 mm, Mann–Whitney ovipositor within the substrate and reinserted it along a different U test, P < 0.001). trajectory. The wasp did not change its body orientation during Deviation from a straight path of the insertion can be esti- an insertion session. A single insertion session contained 1–16 mated by taking the ratio of its arc length and the magnitude of insertions (see Fig. 3B for a typical example of an insertion ses- its R. Comparing the ratios against insertion angles, we see more sion). Individual insertions were not continuous, but consisted insertions with high angles and with low bending in the 2% than of minute retractions and reinsertions, especially when making in the 4% gel (Fig. 3E).

AB C

DE

Fig. 3. Insertion behavior of probing wasps depends on substrate properties. (A) General probing behavior. The wasp positions its ovipositor vertically and punctures the substrate. The sheaths (black arrows) gradually detach and fold away from the ovipositor (white arrow) during deeper insertion. (Scale bar: 5 mm.) (B) Example of a single insertion session in 3D with parameters used in the analysis: R, position vector of the insertion trajectory endpoint; r, radius of the endpoint; d, depth vector; α, insertion angle. (C) Horizontal probing range showing the radii of the endpoints, corrected for the animal orientation (silhouette). Colored circles indicate median values of r for different substrates. Blue, 2% gel; red, 4% gel. (D) Vertical probing range: depth of trajectories plotted against their respective radii. The depth is shallower with increasing radius. (E) Ratio of arc length and the magnitude of R indicates the deviation from a straight path (ratio close to 1 indicates straight insertions). In softer gels, wasps reach higher radii by inserting their ovipositors straight, but at acute angles.

E7824 | www.pnas.org/cgi/doi/10.1073/pnas.1706162114 Cerkvenik et al. Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 oeeto niiulvle psigitrasntdi i.6 Fig. in relative noted little intervals very A (pushing with valves whole, individual a of as movement substrate the into pushed was in also examples (see three 6 the Fig. in presented Steering. are which insertion, and ovipositor Kinematics orientation Valve tip the of 6). peaks Fig. in in seen arrows also (red Protraction be graphs observed. can was how- valves tip valve retracted, ventral dorsal of were the valves of curving ventral no the ever, midline When the ovipositor. was across valve the dorsally dorsal of of curved the valve(s) movements When ventral 5). relative the (Fig. retracted, valves during ventral tip and dorsal ovipositor the the of asymmetry Morphology. Tip Changing speeds only at insertions, were done of were speeds majority curvature, highest low the with although The even curvatures, speed. low low at at observed done always were es i ntegi speetda h oo ftecnor.Tegeneral The contours. S6 ). the (Fig. of bin-size color the the by as effected hardly presented most is is oc- the pattern in the grid points and the of size, in number the equal bin to of dense relative bins points 80 the of into com- (number) divided currence curvature been and has speed axis specific Each of binations. frequency relative the show maps heat (κ 4. Fig. inser- and curvature and between 4 relation (Fig. clear speed a tion also is There gel. speed: insertion (Mann–Whitney gel 2% in faster shown (examples accelerations and (Mann– curvatures gels dimensionless 4% with and 2% the Whitney between distribution curvature in curvature Only tively. values 3E median (Figs. were inser- curvature insertions of of little majority parts very The had directions. however, different consisting tions, in trajectories bends insertion multiple complex of captured even we ally, to (κ up curvature less ( κ) curvature recorded for maximal the values with probing during ovipositors Insertions. their of curve Speed and Curvature ekei tal. et Cerkvenik and

speed (mm/s) speeds low at done generally was insertion ovipositor The utpidwt vpstrdaee)adsifeso h usrt.The substrate. the of stiffness and diameter) ovipositor with multiplied 0.5 1.5 2.5 0 1 2 .Ti ehdwsol bevdi h otr %sub- 2% softer, the in observed only was method This B). pe fpoigdcesswt nraigdmninescurvature dimensionless increasing with decreases probing of Speed .20.04 0.02 0 U hr a lgt u infiat difference significant, but slight, a was There >0.03 %o h at fisrin a dimensionless a had insertions of parts the of ∼1% test, . . . . 1 0.8 0.6 0.4 0.2 0 .I h rtmto,teovipositor the method, first the In S1–S3). Movies 0.0060 0. utpidwt h vpstrwdh.Occasion- width). ovipositor the with multiplied P 73 < dimensionless curvature(-) ms mm ,adw bevdmr insertions more observed we and 0.001), and relative occurrence(-) .Isrin ihhg curvature high with Insertions S6). Fig. <0. −1 eosre la hne nthe in changes clear observed We 02 0.0069 2% n2 e and gel 2% in >0.02 iesols uvtr,wt the with curvature, dimensionless 1.6 ap sdtomtosof methods two used Wasps U ap eeal ostrongly to able were Wasps test, o %ad4 e,respec- gel, 4% and 2% for mm .Wssprobed Wasps S3–S5). Figs. ntedne gel. denser the in .20.04 0.02 0 −1 P or , < ,wt median with 0.001), 0.55 <1.5 0.048 ms mm ms mm n ) Most 4). and dimension- −1 −1 n4% in 4% . eaievleofe eeosre ntepsigmto (Fig. method pushing the the in in 6B observed changes were Minor offset period. valve extended negative an ventral for the protracted having by valves achieved 6A were (Fig. insertions value Curved offset S1). positive Movie a method, around alternating moved the were In valves or valve. method the aligned dorsal the valves pushing of with protraction ovipositors the slight a the using inserting by insertions obtained were Straight methods. these 6 (Fig. method” “alternating the which call process, alternating will insertion clear the we throughout involved valves the method.” method of “pushing common, movements the as more method second, this to The refer will we and strate, bars: (Scale tip. the of the curving of in Protraction result (B) not tip. does 50 arrow) ovipositor to (black leads the valve arrow) of dorsal (white incuving valve(s) and ventral deformation the the of Protraction (A) tip. ovipositor 5. Fig. a bevdi ohgl,soigta hscnas eused be also can this that showing gels, environ- both movements, low-resistance in valve in alternating observed applicable using was method, be second only The might ments. it that ing tip. moved a ovipositor In in of were resulting changed. shape alternatively, changing barely valves moved continuously tip valves the ovipositor the method, the second of pushing, the shape the During and amplitude. together, valve movements relative high valve minimal alternating of with with insertions ovipositor (ii) entire and movements the of pushing (i) relative and valves. substrate ovipositor individual between of interplay movements the from originate and rela- ovipositor. use the a they steer explore that and insert to and to point able motions puncture are valve single tive wasps until a that performed from evidence space been large show have 17, we studies 6, Here, (3, quantitative now. literature no the 48–51), parasitic in mentioned of 18, repeatedly behavior been probing ovipos- has the the wasps Although with done only. through all pierce apparatus is also itor which sometimes (44–47), sub- and integument the host, host’s explore the the and locate pierce 6), to (3, need strate hid- they hosts in Therefore, eggs within. deposit den to substrates into probe wasps Parasitic Discussion piconewtons of Methods hundreds and of order Materials the (SI in were which valves the on 6C (Fig. method alternating the in value offset B A tagtadcre ah eeahee yuigbt of both using by achieved were paths curved and Straight h uhn ehdwsol sdi h otgl indicat- gel, soft the in used only was method pushing The wasps: the by used were insertion ovipositor of methods Two complex are substrate the inside ovipositor the of Movements acting forces net the calculated we data, acceleration the From µm.) and ,weestevle oe rudanegative a around moved valves the whereas S2), Movie rtato fdra n eta avsafc h hp fthe of shape the affect valves ventral and dorsal of Protraction .8s01 0.32s 0.16 s 0.08s 0 s .3 .6 0.086s 0.064 s 0.032s 0 s PNAS and | ulse nieAgs 8 2017 28, August online Published is S3 Figs. – S5 ). A and and C). oi S3 Movie | E7825 and ).

PHYSIOLOGY PNAS PLUS A 5 ventral dorsal 0 0.03 2.5 )

-1 -0.5 0

(mm s 0.02

valve speed -2.5 -1

-5 -1.5 0.2 20 depth (mm) 0.1 0.01 10 -2 0 0 dimensionless curvature (-) (deg) -0.1 -10

offset (mm) -2.5 tip orientation -0.2 -20 0 B 5 *** 0 0.03

2.5 )

-1 -0.5 0

(mm s -2.5 0.02 valve speed -1 -5 0.2 -1.5 20 depth (mm) 0.1 10 0.01 -2 0 0 dimensionless curvature (-) (deg) -0.1 -10

offset (mm) -2.5 tip orientation -0.2 -20 0 C 5 0 0.03 2.5 )

-1 -0.5 0

(mm s 0.02

valve speed -2.5 -1

-5 -1.5 0.2 20 0.1 10 depth (mm) 0.01 -2 0 0 dimensionless curvature (-) (deg) -0.1 -10

offset (mm) -2.5 tip orientation -0.2 -20 0 0 0.5 1 1.5 2 2.5 3 0 0.5 1 time (s) horizontal coordinate (mm)

Fig. 6. Examples of ovipositor insertions. Shown are valve speeds, valve offset, tip orientation, and a 2D trajectory with its curvature (nontracked parts are shown in black). Shaded areas: less accurate calculations due to edge effects. Negative valve offset indicates protraction of dorsal valve(s). (A) Example of a straight insertion in 2% gel showing both methods of insertion: alternating valves (valve speed “out of phase”) and pushing (valve speed “in phase”). Intervals above the graphs indicate the pushing method of ovipositor insertion (curving indicated with asterisks), and red arrows indicate the small changes in tip orientation that occur even on short protractions of the ventral valves (in all graphs). (B) Example of a curved insertion in 2% gel using the pushing method (intervals) with small changes in valve offset. (C) Example of a curved insertion in 4% gel using the alternating valve method. In both B and C, ventral valves are protracted for a prolonged period, which leads to sustained changes in tip orientation, resulting in a curved trajectory.

E7826 | www.pnas.org/cgi/doi/10.1073/pnas.1706162114 Cerkvenik et al. Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 ekei tal. et Cerkvenik mltd ftengtv fstde o lasla oabig- a to probably the inconsistency lead The as always 6). study, (Fig. not orientation our tip does in offset in change ger negative confirmed the partly of only ampli- amplitude curvatures. bigger however, stronger a to was, lead that the will This expect protraction Combining can valve now. ventral one of until point, tude vivo third the in never and species have second any they 64), in 63, observed (36, been hemipterans dead of observed mouth- mouthparts been have in hemipteran properties pro- incurving for the been mechanism Although (35). steering was has parts possible force elements a opposing preloaded as no of posed elements when mechanism Preloaded direction The prob- determined preloading. present. valve, a so-called dorsal in the the toward curved of bend to because dor- tip the ably of the retraction caused (or pronounced valve) valves a sal ventral create the to of protracted con- Protraction are observed, bevel. valves commonly ventral most the both third, asymmetry dition, during the observed the In was other(s), technique methods. the This insertion reinforced. beyond is far tip the valves of more was this or but one effect, ing bending in result (n itself, rarely by an might, which such 1A), create can wasp 62). the 61, which 28, in tip. (27, asymmetrical ways bending reaction three in asymmetric result observed that induce We substrate to hypodermic the of shown from tip been forces bevelled has the which to geometri- similar of of needles, is degrees changes This various shape creating asymmetry. probing 5), by cal the (Fig. achieved of tip observations is ovipositor direct steering the Our that 33). steer- revealed 32, ovipositor process (5, of wasps mechanism in range the ing wide about hypotheses Several postulated a point. been explore puncture single have to a simi- tend from use susbtrate Both the might of probing. hemipterans 59, of and to (35, mechanisms wasps hemipterans similar lar that of are indicates probing 2B) insertion the This for single (Fig. 60). observed a session been within probing has Furthermore, bends what a 37). multiple in the (25, pattern and wasps trajectory that on to insertion studies similar the steering previous of curves in degree multiple a observed of indicates presence insertions The single in methods. insertion both using out- net minimal force. with pushing highly possibly side ani- can substrates, the ovipositors inside of that drill size indicate efficiently small nevertheless the data muscle of Our these of because mals. obtain estimates difficult accurate extremely to making is possible addition, forces not In vivo. currently in is and forces substrate it big- the Unfortunately, substantially probably on ger. are exert substrate the valves exper- within there- the forces mosquito friction can the forces in and actual those valves The with ovipositor iments. compared the the directly are be on study not acting this fore forces in all estimated individ- of forces of The sum 58). movements (55, oscillatory fur- elements the values and ual force to low (55) attributed The puncturing partially proboscis. mosquito are during forces a of Low occur (57) (23). insertion to King ther suggested and Vincent also agree- of in were prediction also is the which with range), (piconewton ment low very are they maxillae that and mandibles (53–56). the axes longitudinal integument, their along host’s oscillate When the our mosquitoes. mouth- feeding in in in slender reported probing seen of been also clearly albeit have (52), movements, is parts alternating behavior of Similar protraction predicted the data. This for allowing valve(s). pulled ovipositor, other being the are anchors valves which some at mechanism, the inser- ovipositor to predicts for According used which tion. be (23), can movement King the valve alternating and with that Vincent accordance by in proposed is mechanism This environments. high-resistance in is,temrhlg fteoioio i saymti (Fig. asymmetric is tip ovipositor the of morphology the First, obtained were trajectories ovipositor curved that observed We revealed S3–S5) (Figs. valves the on forces net of Estimation )osre noreprmns eod yprotract- by Second, experiments. our in observed 2) = neto u oicesdfito ewe h vpstrand ovipositor the between ovipositor friction for increased required to energy of due amount insertion the increased also tor properties we material fruits. densities, different in to gel adapt different can we wasps in that that Because kinematics hypothesize this is probing well. in in estimation reasonably changes Our conditions used saw such fruit. gels represent decaying the gels the the of of those properties match deter- physical to study is impossible the thus is it whether with It known. mine and infested not are condition, fruits fruits rele- decaying the one healthy in vant of properties only decrease a mechanical Unfortunately, drastically of was larvae. val- fruit-fly they reported estimates gel The that are densest fruits. expected fruits The these for than 73). the softer ues and (72, in magnitude 71) fruits of (70, used order peaches citrus gel mature of softest smaller for magnitude the peel reported of moduli of orders The the two S1) fibers. than approximately (Fig. the and were study moduli as layers this of fruits, elastic composed the and and and shear anisotropic gels are the some latter them are between There substituted densities. differences visualize we different to important so of fruits, gels impossbile substrates, gellan citrus such is translucent for in others, It process amongst (67–69). probing apples are, the and hosts figs, for the peaches, denser into substrates into natural probe penetration The wasps makes costly. which energetically more 66), increase substrates (65, stress failure density and gel ) S1 with (Fig. Stiffness speed. insertion on gel. stiffer the of experienced result This forces a reaction crucial. as stronger are the valves by the enhanced probing curva- of probably the is movements their adjusting relative by enlarge which achieved to for is this ture ovipositor that the show forces data of Our lateral space. bending wasps large substrates, use stiff avoid to in Therefore, need to buckling. in surface result might the that to perpendicular itor curvatures. high very require opposite the would in that probe because to such direction, possibility However, the angles. limits insertion shallow ones, angular stiff at an in puncture than to smaller wasps are the forces allowing these buck- when in substrates, result soft possible might In only forces ling. our lateral is large with because angles puncturing small, acute are of forces at the time Puncturing the between setup. at angle camera substrate the the determine and to Unfor- ovipositor impossible surface. substrate was the it to respect ovipos- tunately, with their angles inserted dis- acute wasps at horizontal the itors because large be still a may were with This insertions placement. of insertions majority including the sub- straight, gel, Despite the soft substrate. nearly the on soft and in the dependent radius in ovipositor is larger larger a the cone slightly the is of and of length stiffness radius strate the The to base. equal curved a height the the with within cone tip its or ovipositor only the the rotating ani- body, substrate. the of that the capable is of are range observed position mals the that the for Considering explanation change possible direction. not other one does in animal bending the a from explain described direction only mechanisms for any steering above in beneficial However, steered therefore point. be is puncture can the single ovipositor it arguably the and if is probing, wasp orienta- Puncturing the in body point. step single puncture difficult a single most from a above. this using do described tion, can mechanisms wasp steering the and Furthermore, insertion the recordings. using our nec- of was focus which of but depth valves, the ventral increase to two distinction essary the no between which made in be images shadow can of analysis the from arises iia oicesdsbtaedniy uvn fteoviposi- the of curving density, substrate increased to Similar effect negative a has density substrate that showed also We ovipos- the position to need larger a is there gel, stiff the In a to similarly envisioned be can range the species, our For by volume large a explore can wasps that show results Our PNAS | ulse nieAgs 8 2017 28, August online Published | E7827

PHYSIOLOGY PNAS PLUS the substrate. This explains the lower insertion speeds for curved Experimental Setup and Data Acquisition. The 3D insertion path of the parts of trajectories observed in our study. It also indicates that ovipositor in the substrate was recorded with two synchronized high-speed curving is an energetically expensive behavior that might be bet- video cameras (Fastcam SA-X2; Photron) fitted with macro lenses (MP-E 65; Canon; 5× magnification factor) at 125 fps. The cameras’ optical axes were ter avoided if possible. ◦ We show that reciprocal valve movements are used when perpendicular to each other (89 ± 1 ; Fig. 7A). Two near-collimated light beams were produced by using combinations of an approximate point light inserting slender probes into solid substrates. The estimated net source and a 4-diopter lens, arranged in a backlight configuration. This forces acting on the valves are the first quantification of the push– resulted in a shadow image and a depth of focus of ∼1 mm. The cameras pull mechanism in vivo. The low values are in agreement with recorded probing events in a cuvette (inner dimensions 10.5 × 10.5 × 6 mm) the proposed insertion mechanism characterized by minimal net with the probing medium in the center. A micromanipulator allowed trans- external pushing force. Furthermore, the relative position of the lation of the cuvette in three directions to get the ovipositor within the field valves dictates the shape of the tip and influences the direction of view and the depth of field of the cameras. The cuvette was located in a of probe insertion, probably by manipulating the size and direc- closed-off glass arena (inner dimensions 48 × 48 × 48 mm). tion of the substrate reaction forces. This improves the insight in Conversion of camera image pixels to the actual distances was done by the overall mechanism of oviposition in hymenopterans. Under- using still images of copper specimen support grids for electron microscopy (0050-Cu; Electron Microscopy Sciences). The grid mesh size was measured standing of the mechanism will deepen the knowledge of adapta- with a calibrated microscope (Leica M205FA; Leica Microsystems; 20× mag- tions in the ovipositor apparatus and the evolution of the taxon nification), after which the grids were put in a cuvette and embedded in the as a whole. gel, which was also used in the experiments (see below). The difference in In addition, our findings can help advance the development of vertical position of the cameras was corrected based on the mean vertical steerable man-made probes that is on the rise in the past decade coordinate (depth) of the most distal part of the tracked ovipositor tip in (27, 62, 74) and is particularly relevant for the design of multiele- both camera views. ment probes (39, 40, 75). Miniaturization of the needle diameter An additional camera (piA640-210-gm, 50 fps; Basler AG; with a Nikkor should presumably be possible also for the needles used in solid AF-50 lens; Nikon) recorded the spatial orientation of the probing wasp substrates if the individual elements are operated in an alternat- from above. The conversion from pixels to actual lengths for the images of this camera was calculated based on the known dimensions of the cuvette. ing manner. The adjustable bevel shape, although already par- Image time stamps were used to match the recordings between nonsynchro- tially implemented (e.g., ref. 61), can be greatly enhanced by nized cameras. implementing pretension of the elements. Despite having mul- Probing behavior was recorded (Fig. 7A) in substrates with two different tiple elements, the control of such needles may not necessarily densities to assess the effect of substrate properties. The substrate contained be complicated, as it would be enough to monitor the juxtaposi- commercially available apple juice (10%), tap water (90%), and gelling tion of individual elements that would dictate the direction and agent (Phytagel; Sigma-Aldrich) in either 0.02 g mL−1 (2%) or 0.04 g mL−1 amplitude of curving. (4%). The bottom of each cuvette contained a live, 4- to 7-d-old C. capitata larva and some of its rearing medium, covered with a fine cloth. A piece Materials and Methods of wet filter paper with thinly spread larval medium was on the top of the Substrate Properties. We used gellan gel, a microbial polysaccharide (76), substrate. The presence of these cues induced and prolonged the probing as the probing substrate because of its translucency, isotropic properties, behaviors of the wasps. At the start of an experiment, several wasps were and readiness of the animals to insert their ovipositors in it. The rheo- put in the arena, and nonresponsive females (i.e., those not motivated to logical properties, namely, the storage (G0) and loss (G00) moduli, of the probe) were removed until only one responsive animal was left. Upon initi- gels used in the experiments were measured with a rotational rheometer ation of probing, the position of the arena was manually adjusted to have (Anton Paar) and a cone plate probe (diameter of 25 mm) at 20◦C. The the ovipositor in focus, after which no further adjustments were made to gels were amorphous and considered isotropic (i.e., random cross-linking of avoid movement artefacts. After a successful recording, wasps were isolated hydrophilic polymer chains was assumed). To ensure a strong contact with to ensure analysis of probing behavior of each animal in both 2% and 4% the equipment, the gels were poured onto the base plate, and the probe substrate. Each gel was used for several wasps until either the gel started was lowered into the gels while they were still warm. The gel was then left showing signs of drying or the transparency reduced too much due to the to cool down before starting the experiments. The linear strain region of number of probing paths. the gel was obtained by first measuring the response to a changing strain (amplitude range: 0.1–100%) at a constant angular velocity (0.16 rad s−1). Another gel of the same concentration was used for the measurements at a constant strain within the linear strain regime (0.2%) with a changing AB angular frequency of the probe (1–100 rad s−1). The frequency measure- high-speed ments were repeated three times with 1-min intervals. The obtained moduli top cameras were averaged across repetitions and frequencies to calculate the dynamic camera shear modulus (G∗) and the dynamic elastic modulus (E∗). We assumed a Poisson’s ratio (ν) of 0.5 because of the high water content of the gels. See offset SI Materials and Methods for details of the calculations.

Animals. Adult parasitic wasps (D. longicaudata) and their host, the Medi- terranean fruit-fly Ceratitis capitata) were kept separately in rearing cages (30 × 30 × 30 cm, BugDorm; MegaView Science) at 24 ◦C and 12/12-h arena v light/dark cycle, with ad libitum access to water and food. Wasp were fed (animals) tangent v t commercially available honey, and flies were fed a mixture of glucose and lens brewer’s yeast dissolved in tap water. point source final The flies could freely reproduce and lay their eggs in perforated plas- path tic bottles. The eggs were harvested and put in oxygenated water for 1 d before transferring them to the larval medium, which was a mixture of Fig. 7. Experimental setup and calculation of insertion speed. (A) Two high- brewer’s yeast, carrot powder, sodium benzoate (food preservative), methyl- speed cameras were positioned perpendicular to each other, each aligned paraben (antifungal agent), hydrochloric acid (HCl; acidity regulator), and with a near-collimated light beam (yellow). The arena with the substrate- tap water. The larvae were exposed to parasitoids between the fourth and filled cuvette and the animals was located in the field of view of both cam- seventh day after hatching. After parasitation, the larvae were taken away eras. The third camera was located above the experimental arena, directed from the wasps and left to develop and pupate in small boxes of vermiculite. downward. (B) Schematics of the ovipositor during insertion in 2D (only After ∼2 wk, the wasps emerged from pupae of parasitized larvae. Fly lar- two valves are shown). The tangential projection (vt) of the valve veloc- vae used for propagation of fly colonies were never exposed to parasitation ity (v) along the final centerline (dashed, red line) was taken as the inser- and were kept apart from the wasps. For details of the rearing protocol, see tion speed. Insertion speed was calculated from the velocity of the fore- SI Materials and Methods. most valve.

E7828 | www.pnas.org/cgi/doi/10.1073/pnas.1706162114 Cerkvenik et al. Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 rlfruafraprmtial endcre(77): curve defined parametrically gen- a the from for obtained formula expression eral the using by curvature calculated the was tor of amplitude the trajectories, the ekei tal. et tip Cerkvenik ovipositor the of part distal most the of data position filtered multiplying (30 ovipositor by cur- obtained dimensionless The was length. arc vature the to and respect (s), with derivatives length arc its of tion where positive the with aligned was axis body the to set was the insertion first origin the tra- that common 3D such All aligned curvature. were the jectories calculating for a sec- needed continuous derivatives ensure into to ond function cameras spline were quintic trajectories a the 3D with smoothed The of system. coordinate systems world right-handed coordinate perpendicular two below). (defined system coordinate the world with the head the of animal’s of the axis angle toward direction directed the axis by major orientation defined ellipse’s The was frame. animal each the of in was (azimuth) animals were animal segmented Ellipses to segmentation. the open fitted the then morphological of improve to by individ- silhouette operations followed close the the and was to that This adapted recognizable. so clearly thresholds sequence on image based ual images binary to to up within centerline ovipositor tip the segmented ovipositor the of the of extrapolation boundary of for the linear part used distal by not was most determined skeleton the was the of of position end irregu- The distal to fitting. the due gel, irregular tip the often the in was Because larities (good- ovipositor inspection). visual skeletonized it by the through determined of curve was spline fit smoothed of cubic ness a ovipos- fitting segmented the and skeletonizing itor by obtained was frame each 1.1). Version Toolkit; Segmentation (ilastik: and ilastik Learning with the Interactive done of was images segmentation background The the trajectory. in ovipositor complete a other from obtain with segmented to combined be then images to and had separately frames images five background first the that the sup- ovipositor in the sometimes only of visible section procedure, was This erosion input. manual and with dilation plemented were threshold a artefacts and a by objects silhou- removed on Smaller ovipositor recognizable. the based clearly that was images such ette sequence binary fol- image was to each to This conversion adapted images. a all from by subtracted frames lowed was five first insertion) of each (average of background the First, R2016b; MathWorks). and R2013a (Versions Matlab in code custom-built using insertion Image full the inser- point. only individual contained puncture trajectory. they into that the such split cropped ovipos- were and from tions the sessions sev- away insertion of of one of substrate movement sequences consisted as as the general defined surface into in we the itor which session of ovipositor insertions, a point the eral Such puncture of session. single movements insertion all a defined through We made once. than more Tracking. Automatic Analysis Data κ rbn eoiywscluae ytkn h rdeto the of gradient the taking by calculated was velocity Probing the combining by obtained were trajectories probing 3D The converted and cropped were camera top the from Images in ovipositor the of centerline the images, cleared the using By by segmented were ovipositors the sequences, image these In Z = = xst orc o h retto fteaiass htthe that so animals the of orientation the for correct to axis s l(s) k l(s) (y k stevco ucino h uv xrse sfunc- as expressed curve the of function vector the is 0 l(s) 0 z × 00 ,etmtdfo E images). SEM from estimated µm, − 0 l(s) (0, k y 3 00 00 0, igeaia sal rbdtesubstrate the probed usually animal single A z k 0 h uvswr hnrttdaround rotated then were curves The 0). ) 2 (x + (x 0 2 0 00 and + z 0 y − 0 00 2 x κ + eoetefis n second and first the denote 0 z ihtedaee fthe of diameter the with Y z 00 0 ) 2 2 xs o ahpitof point each For axis. ) ( + 3 x κ 0 y >7.5 fteoviposi- the of 00 − x ◦ . 00 y 0 ) 2 , Y ple wc yuigteMta lfitfnto)fo h direc- the from ( function) data filtfilt tion Matlab the using by twice applied the orienta- (2 baseline into tion the reference subtracting of by reference frame of image frame overall the ovipositor from the data retained orientation S7 but the (Fig. artefacts, characteristics frame to temporal frame the reduced data 18.75 orientation the (2 filtered time We oviposi- in plane. tip. the horizontal valve of the dorsal orientation and the the tip end of between tor the length angle of the the length of calculated The one-third centerlines We centerline. as the taken the of was part part end of the from extrapolation lated ends spurious the remove insertion and to the need skeletons above). of (as no the in was tracking back- there Automated routine of complex so using images. a segmentation accurate, by to the was the done due of was example, results ground which unsatisfactory latter 6C, yielded the Fig. Matlab in In shown ilastik. example the for the of protraction the denotes valve. negative ventral and foremost valve, protraction as dorsal denotes taken offset the was Positive of frame valves. digi- each the in the between tips offset between valve the the distance of valves ends the the distal addition, most of tized In velocity tangential above. the described calculate as to used were lines valve its on dorsal based The valves 1A). ventral focus. (Fig. the morphology in from distinguished was dorsal be tip the easily all could ovipositor in of digitized the manually ends where were distal tips frames valve most ventral foremost The the ventral recognized. and sec- (foremost) be the protruded of could most view valves the valve the the only of in so silhouettes curve discernible, only images, were not backlit did We our (i.e., In insertions. within camera). DoF of ond moving the parts was of only ovipositor plane the to the where and track- sequences view the image camera limited chose single which (DoF), a field to of ing depth narrow a in Kinematics. sulted Valve of Analysis dorsal the of speed valve. ventral the foremost between each the discriminated and in veloc- centerline also instantaneous ovipositor We the instantaneous frame. of the to components tangential the ity as of calculated (see were magnitude speeds focus the retraction in and clearly insertion were Valve valves below). individual where examples ovipositor. tion entire the of valve speed foremost insertion the the of as 7B). speed (Fig. taken point The was time analyzed. each not at path were final Retractions the tangen- of vector centerline velocity the the to of tial component the of magnitude the as a using effectively function, 4 “filtfilt” Matlab using twice applied (2 rmrf 8 o eal ffrecluain see calculations force Methods. of details For 78. ref. from dryad.8bc95 ipie yidia oe fteoioio suigsimple assuming ovipositor S8 (Fig. the properties of material taking model with by combined cylindrical data, estimated simplified position a were filtered the S3–S5) of (Figs. derivative second valves Valves. the the the on on Forces forces Net net the and Accelerations the Estimating th nd retto fteoioio i uigisrinwscalcu- was insertion during tip ovipositor the of Orientation except above, described as segmented was ovipositor The center- frame-by-frame the with together points digitized The inser- three for done was 2D in kinematics valve of Analysis taken was speed insertion the dataset, 3D the of analysis the In h aaaeaalbeo ra (http://dx.doi.org/10.5061/ Dryad on available are data The re filter). order re o-asBtewrhfitr uoffrequency cutoff filter, Butterworth low-pass order zapidtieuigteMta lfitfnto) which function), filtfilt Matlab the using twice applied Hz nd re o-asBtewrhfitr uoffrequency cutoff filter, Butterworth low-pass order nd i.S7 Fig. ). re o-asBtewrhfitr uoffrequency cutoff filter, Butterworth low-pass order D–F PNAS ). .Tedniyo h uil a taken was cuticle the of density The ). | ulse nieAgs 8 2017 28, August online Published h ihmgicto sd re- used, magnification high The .W hntransformed then We A–C). IMtrasand Materials SI | 15 E7829 1 The Hz Hz

PHYSIOLOGY PNAS PLUS ACKNOWLEDGMENTS. We thank Francisco Beitia (Instituto Valenciano de Cees Voesenek for advice on analytical methods; Dimitra Dodou (Delft Investigaciones Agrarias) for providing parasitoids and Carlos Caceres´ (Inter- University of Technology) for advice on the experiments; and the user national Atomic Energy Agency) for providing fruit-flies for our colony; committee of the Netherlands Organization for Scientific Research Divi- Marleen Kamperman and Marco Dompe´ (Physical Chemistry and Soft sion Applied and Engineering Sciences (NWO TTW) WASP project and Matter, Wageningen University and Research) for their assistance with the members of the research group for their useful discussions. This the rheological measurements of the substrates; Henk Schipper, Remco work was supported by NWO domain applied and engineering sciences Pieters, and Karen Leon-Kloosterziel for technical support and animal care; (NWO TTW).

1. Le Lannic J, Nenon´ JP (1999) Functional morphology of the ovipositor in Megarhyssa 29. Le Ralec A, Rabasse JM, Wajnberg E (1996) Comparative morphology of the ovipos- atrata (Hymenoptera, Ichneumonidae) and its penetration into wood. Zoomorphol- itor of some parasitic Hymenoptera in relation to characteristics of their hosts. Can ogy 119:73–79. Entomol 128:413–433. 2. Ovruski S, Aluja M, Sivinski J, Wharton R (2000) Hymenopteran parasitoids on fruit- 30. Gerling D, Quicke DLJ, Orion T (1998) Oviposition mechanisms in the whitefly para- infesting Tephritidae (Diptera) in Latin America and the Southern United States: sitoids Encarsia transvena and Eretmocerus mundus. BioControl 43:289–297. Diversity, distribution, taxonomic status and their use in fruit fly biological control. 31. Ko SY, Frasson L, Baena FRY (2011) Closed-loop planar motion control of a steerable Integrated Pest Manag Rev 5:81–107. probe with a “programmable bevel” inspired by nature. IEEE Trans Robot 27:970– 3. Belshaw R, Grafen A, Quicke DLJ (2003) Inferring life history from ovipositor mor- 983. phology in parasitoid wasps using phylogenetic regression and discriminant analysis. 32. Quicke DLJ, Fitton M, Harris J (1995) Ovipositor steering mechanisms in braconid Zoolog J Linn Soc 139:213–228. wasps. J Hymenoptera Res 4:110–120. 4. Smith EL (1970) Evolutionary morphology of the external insect genitalia. 2. 33. Quicke DLJ, Fitton MG (1995) Ovipositor steering mechanisms in parasitic wasps of Hymenoptera. Ann Entomol Soc Am 63:1–27. the families Gasteruptiidae and Aulacidae (Hymenoptera). Proc R Soc Biol Sci 261:98– 5. Quicke DLJ, Fitton MG, Tunstead JR, Ingram SN, Gaitens PV (1994) Ovipositor struc- 103. ture and relationships within the Hymenoptera, with special reference to the Ichneu- 34. Quicke DLJ (1991) Ovipositor mechanics of the braconine wasp genus Zaglyptogastra monoidea. J Nat Hist 28:635–682. and the ichneumonid genus Pristomerus. J Nat Hist 25:971–977. 6. Ghara M, Kundanati L, Borges RM (2011) Nature’s Swiss army knives: Ovipositor struc- 35. Pollard DG (1969) Directional control of the stylets in phytophagous Hemiptera. Proc ture mirrors ecology in a multitrophic fig wasp community. PLoS One 6:e23642. R Entomol Soc Lond Ser A Gen Entomol 44:173–185. 7. Schudder GGE (1961) The comparative morphology of the insect ovipositor. Trans R 36. Pollard DG (1971) The use of polyporus for the investigation of stylet behaviour in Entomol Soc Lond 113:2–40. the Hemiptera. Entomol Exp Appl 14:283–296. 8. Lyngnes R (1960) Shape and function of the ovipositor in the three hymenopter- 37. Compton S, Nefdt R (1988) Extra-long ovipositors in chalcid wasps: Some examples ous species: Ephialtes extensor Thom. (Ichneumonidae), Spathius exarator L. (Bra- and observations. J Antenn 12:102–105. conidae), and Plutothrix coelius walk. (Chalcididae). Norsk Entomologisk Tidsskrift 38. Sears P, Dupont P (2006) A steerable needle technology using curved concentric tubes. 11:122–134. Proceedings of the 2006 IEEE/RSJ International Conference on Intelligent Robots and 9. King PE (1962) The muscular structure of the ovipositor and its mode of function in Systems, Beijing (IEEE, Piscataway, NJ), pp 2850–2856. Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae). Proc R Entomol Soc Lond 39. Parittotokkaporn T, et al. (2009) Soft tissue traversal with zero net force: Feasibility Ser A Gen Entomol 37:121–128. study of a biologically inspired design based on reciprocal motion. Proceedings of 10. Quicke DLJ (2015) The Braconid and Ichenumonid Parasitoid Wasps: Biology, Sys- the 2008 IEEE International Conference on Robotics and Biomimetics, Bangkok (IEEE, tematics, Evolution and Ecology (John Wiley & Sons, Ltd, Chichester, UK), 1st Ed, Piscataway, NJ), pp 80–85. p 595. 40. Frasson L, et al. (2010) STING: A soft-tissue intervention and neurosurgical guide to 11. Vilhelmsen L, Turrisi GF (2011) Per arborem ad astra: Morphological adaptations to access deep brain lesions through curved trajectories. Proc IME H J Eng Med 224: exploiting the woody habitat in the early evolution of Hymenoptera. Arthropod 775–788. Struct Dev 40:2–20. 41. Roesthuis RJ, van Veen YR, Jahya A, Misra S (2011) Mechanics of needle-tissue inter- 12. Vilhelmsen L (2000) Before the wasp-waist: Comparative anatomy and phylogenetic action. Proceedings of the Proceedings of the 2011 IEEE/RSJ International Confer- implications of the skeleto-musculature of the thoraco-abdominal boundary region ence on Intelligent Robots and Systems, San Francisco (IEEE, Piscataway, NJ), pp 2557– in basal Hymenoptera (Insecta). Zoomorphology 119:185–221. 2563. 13. Sharkey MJ (2007) Phylogeny and classification of Hymenoptera. Zootaxa 1668:e548. 42. Ko SY, Baena RYF (2012) Trajectory following for a flexible probe with state/input 14. Davis RB, Baldauf SL, Mayhew PJ (2010) The origins of species richness in the constraints: An approach based on model predictive control. Robot Autonom Syst Hymenoptera: Insights from a family-level supertree. BMC Evol Biol 10:109. 60:509–521. 15. Peters RS, et al. (2017) Evolutionary history of the Hymenoptera. Curr Biol 27: 43. Elgezua I, Kobayashi Y, Fujie MG (2013) Survey on current state-of-the-art in needle 1013–1018. insertion robots: Open challenges for application in real surgery. Proced CIRP 5:94–99. 16. Matushkina N, Gorb S (2007) Mechanical properties of the endophytic ovipositor in 44. Consoli FL, Kitajima EW, Postali Parra JR (1999) Sensilla on the antenna and ovipositor damselflies (Zygoptera, Odonata) and their oviposition substrates. Zoology (Jena) of the parasitic wasps Trichogramma galloi Zucchi and T. pretiosum Riley. Microsc Res 110:167–175. Tech 45:313–324. 17. Polidori C, Garc´ıa AJ, Nieves-Aldrey JL (2013) Breaking up the wall: Metal-enrichment 45. Hawke SD, Farley RD, Greany PD (1973) The fine structure of sense organs in the in ovipositors, but not in mandibles, co-varies with substrate hardness in gall-wasps ovipositor of the parasitic wasp, Orgilus lepidus Muesebeck. Tissue Cell 5:171–184. and their associates. PLoS One 8:e70529. 46. van Lenteren JC, et al. (2007) Structure and electrophysiological responses of gusta- 18. Kundanati L, Gundiah N (2014) Biomechanics of substrate boring by fig wasps. J Exp tory organs on the ovipositor of the parasitoid Leptopilina heterotoma. Arthropod Biol 217:1946–1954. Struct Dev 36:271–276. 19. Smith EL, Behavior O (1972) Biosystematics and morphology of Symphyta - III exter- 47. Shah ZA (2012) Morphology, ultrastructure, and probable functions of the sense nal genitalia of Euura (Hymenoptera: Tenthredinidae): Sclerites, sensilla, musculature, organs on the ovipositor stylets of the hymenoptran parasitoid, Venturia canescens development and oviposition behaviour. Int J Insect Morphol Embryol 1:321–365. (Gravenhorst). Microsc Res Tech 75:876–883. 20. Field SA, Austin AD (1994) Anatomy and mechanics of the telescopic ovipositor system 48. van Lentern JC, et al. (1998) Functional anatomy of the ovipositor clip in the parasitoid of Scelio latreille (Hymenoptera: Scelionidae) and related genera. Int J Insect Morphol Leptopilina heterotoma (Thompson) (Hymenoptera: Eucolidae), a structure to grip Embryol 23:135–158. escaping host larvae. Int J Insect Morphol Embryol 27:263–268. 21. Vilhelmsen L, Isidoro N, Romani R, Basibuyuk HH, Quicke DLJ (2001) Host location and 49. Vinson SB (1976) Host selection by insect parasitoids. Annu Rev Entomol 21:109–133. oviposition in a basal group of parasitic wasps: The subgenual organ, ovipositor appa- 50. Spradbery JP (1970) Host finding by Rhyssa persuasoria (L.) in ichneumonid parasite ratus and associated structures in the Orussidae (Hymenoptera, Insecta). Zoomorphol- of siricid woodwasps. Anim Behav 18:103–114. ogy 121:63–84. 51. Ghara M, Ranganathan Y, Krishnan A, Gowda V, Borges RM (2014) Divvying up an 22. Heatwole H, Davis DM, Wenner AM (1962) The behaviour of Megarhyssa, a genus of incubator: How parasitic and mutualistic fig wasps use space within their nursery parasitic hymenopterans (Ichneumonidae: Ephialtinae). Z Tierpsychol 19:652–664. microcosm. Arthropod Plant Interact 8:191–203. 23. Vincent JFV, King MJ (1995) The mechanism of drilling by wood wasp ovipositors. 52. Kim BH, Kim HK, Lee SJ (2011) Experimental analysis of the blood-sucking mechanism Biomimetics 3:187–201. of female mosquitoes. J Exp Biol 214:1163–1169. 24. Vilhelmsen L (2003) Flexible ovipositor sheaths in parasitoid Hymenoptera (Insecta). 53. Gordon RM, Lumsden WHR (1939) A study of the behaviour of the mouth-parts of Arthropod Struct Dev 32:277–287. mosquitoes when taking up blood from living tissue together with some observations 25. Elias LG, Teixeira SP, Kjellberg F, Santinelo Pereira RA (2012) Diversification in the use on the ingestion of microfiliarie. Ann Trop Med Parasitol 33:259–278. of resources by Idarnes species: Bypassing functional constraints in the fig-fig wasp 54. Griffiths RB, Gordon RM (1952) An apparatus which enables the process of feeding by interaction. Biol J Linn Soc 106:114–122. mosquitoes to be observed in the tissues of a live rodent; together with an account 26. Abolhassani N, Patel R, Moallem M (2007) Needle insertion into soft tissue: A survey. of the ejection of saliva and its significance in malaria. Ann Trop Med Parasitol 46: Med Eng Phys 29:413–431. 311–319. 27. Misra S, Reed KB, Douglas AS, Ramesh KT, Okamura AM (2008) Needle-Tissue inter- 55. Kong X, Wu C (2009) Measurement and prediction of insertion force for the mosquito action forces for bevel-tip steerable needles. Proc Biennial IEEE/RAS-EMBS Int Conf fascicle penetrating into human skin. J Bionic Eng 6:143–152. Biomed Robot Biomechatron 2:224–231. 56. Choumet V, et al. (2012) Visualizing non infectious and infectious Anopheles gambiae 28. Abayazid M, Roesthuis RJ, Reilink R, Misra S (2013) Integrating deflection models blood feedings in naive and saliva-immunized mice. PLoS One 7:e50464. and image feedback for real-time flexible needle steering. IEEE Trans Robotics 29: 57. Kong XQ, Wu CW (2010) Mosquito proboscis: An elegant biomicroelectromechanical 542–553. system. Phys Rev E Stat Nonlin Soft Matter Phys 82:011910.

E7830 | www.pnas.org/cgi/doi/10.1073/pnas.1706162114 Cerkvenik et al. Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 0 epl A rea P uke S esnD 20)Muhatmrhlg and morphology Mouthpart (2003) DR Nelson JS, Buckner TP, Freeman RA, Leopold 60. penetration Stylet (2001) imitating TJ Henneberry microneedles CC, Chu DR, jagged Nelson JS, harpoonlike Buckner TP, Freeman Combined 59. (2008) al. et H, Izumi 58. 5 agK,Vee T irsu J aeoT otelI 18)Aa-iepolysac- Agar-like (1982) IW Cottrell T, Kaneko PJ, Mirrasoul GT, Veeder KS, Kang 65. mouth- the of characterization mouthparts Morphological (2010) the Y Zhang of C, Zhang morphology W, Dai External L, Zhao (2014) C 64. Zhang L, Jin Y, Lu Nonholonomic L, (2006) Pan AM W, Okamura Dai GS, Chirikjian 63. NJ, Cowan JS, Kim IIIRJ, Webster 62. of evaluation Experimental (2011) RYF Baena G, Dogangil SY, Ko F, Ferroni L, Frasson 61. 7 inyEJ(97 yai lsi rpriso oefut uiggot n devel- and growth during fruits some of properties elastic Dynamic (1967) EEJ Finney compres- uniaxial by 67. gels gellan of Characterization (1990) al. et AMMA, Nussinovitch 66. ekei tal. et Cerkvenik tltpntaino otpat ytegas-igdsharpshooter, glassy-winged the by plants host coagulata of penetration stylet America Soc by vibration. with experiment insertion Eng Electron its and proboscis mosquito’s nio Microbiol Environ a by produced charide leafhopper Micron vector the of parts sensilla. the to reference special planthopper whitebacked the of steering. needle of modeling nature. by inspired tip bevel programmable a with Surg probe steerable novel a opment. creep. and relaxation stress sion, eii argentifolii Bemisia 6:189–197. 41:754–759. gi n Res Eng Agric J Hmpea Cicadellidae). (Homoptera: 94:761–768. 3:425–431. 43:1086–1091. Hmpea lyoia)it otla tissue. leaf host into Aleyrodidae) (Homoptera: Pseudomonas 12:249–256. n oo Res Robot J Int smoetxstriatus Psammotettix Micron etr Stud Texture J oael furcifera Sogatella rhoo tutDev Struct Arthropod pce:Pouto n ai properties. basic and Production species: 56:8–16. 25:509–525. 21:37–50. Hmpea epaia) with Delphacidae), (Hemiptera: L)(eitr:Cicadellidae). (Hemiptera: (L.) 32:189–199. EJTasElectr Trans IEEJ n Entomol Ann Homalodisca Robot J Appl 8 ev L rwigH,Glta E(91 feto otfutseis ie n color and size, species, fruit host of Effect (1991) FE Gilstrap HW, Browning JL, Leyva 68. 8 icn F,WgtUK(04 einadmcaia rpriso netcuticle. insect of properties mechanical and Design extracellu- (2004) UGK an Wegst JFV, gum, Vincent 78. gellan of (1966) studies B O’Neal Structural 77. (1983) L Bengt PE, “pro- a Jansson with 76. steering needle Two-dimensional (2010) RYF Baena BL, flexible Davies of SY, Mechanics (2010) Ko AM Okamura 75. KT, Ramesh BW, Schafer KB, Reed S, Misra peel orange 74. of properties physico-mechanical Post-harvest (2006) BS Reddy peel KK, Singh orange of 73. properties Mechanical (2002) FS Davies AA, Teixeira MW, Fidelibus anal- by 72. firmness peach of Determination (1987) SV Bowers T, behavior McDonald elastic MJ, of Delwiche aspects Some 71. (1968) PA Adrian JW, Rumsey RA, Bradley RB, Fridley 70. the in preference habitat host Innate (2016) MM Viscarret AL, Nussenbaum DF, Segura 69. longicaudata fslce fruits. selected of learning. through parasitoid nprstzto of parasitization on rhoo tutDev Struct Arthropod 422. p Ed, 1st elodea. Pseudomonas by 139. elaborated polysaccharide lar Syst Robots preliminaries. Intell Modeling nature: by inspired bevel” grammable tissue. soft through steered robotically needles fruit. and acid. gibberellic 1062. with pre-harvest treated fruit and forces. impact of ysis odEng Food J ucinlsgicneadmodifications and significance Functional longicaudata: Diachasmimorpha Hmnpea Braconidae). (Hymenoptera: lmnayDfeeta Geometry Differential Elementary 2319–2324. rn mScArcEng Agric Soc Am Trans LSOne PLoS nsrpaludens Anastrepha rn mScArcEng Agric Soc Am Trans 73:112–120. 33:187–199. PNAS 11:e0152222. | ulse nieAgs 8 2017 28, August online Published Dpea ehiia)by Tephritidae) (Diptera: 11:46–49. nio Entomol Environ 30:249–254. n oo Res Robot J Int dONa Aaei,London), (Academic, B O’Neal ed , rn mScArcEng Agric Soc Am Trans abhd Res Carbohydr 20:1469–1474. 00IE/S n Conf Int IEEE/RSJ 2010 29:1640–1660. Diachasmimorpha | 45:1057– 124:135– E7831

PHYSIOLOGY PNAS PLUS