Descriptions of immature stages of four species of the genera Graptus, Peritelus, Philopedon, and Tanymecus and larval instar determination in Tanymecus (Coleoptera, Curculionidae, Entiminae) Author Gosik, Rafal Author Sprick, Peter Author Morris, Michael G. text ZooKeys 2019 813 111 150 http://dx.doi.org/10.3897/zookeys.813.30336 journal article http://dx.doi.org/10.3897/zookeys.813.30336 1313-2970-813-111 AF1D1300BD4B4C1795B48567EFAD850C AF1D1300BD4B4C1795B48567EFAD850C Tanymecus palliatus Specimens examined. 3 mature larvae: Germany, Brandenburg, Cottbus: Kittlitz, collected on 09.08.2011 in a permanent field of Medicago sativa L., together with a Tanymecus pupa and many larvae of Otiorhynchus ligustici (Linnaeus, 1758). 12 first instar larvae: A female collected in the field (Kittlitz) laid eggs on 17.05.2012 in the laboratory in Hannover. Larvae, hatched from eggs on 31.05.2012, were used for this study. Description of the mature larva. Body length: 8.3-10.0 mm, body width at the widest part (level of first abdominal segment): 2.5-3.2 mm, head width: 1.6-1.8 mm, head height: 1.4-1.6 mm. Body (Figs 10-12). Moderately stout, slightly curved, rounded in cross section. Prothorax slightly smaller than mesothorax; metathorax as wide as mesothorax. Prothorax with a pair of dark sclerotized, conical protuberances, placed dorsally, near margin with mesothorax (Fig. 59). Spiracles (of thoracic and abdominal segments 1-8) annular. Abdominal segments 1-7 of almost equal length, segment 8 wide, flattened posteriorly, with conical lateral lobes. Abdominal segment 9 strongly reduced, consisting of 4 well-isolated lobes, of which the lateral lobes are the biggest; segment 10 consists of 4 anal lobes of various size. Anus located ventrally (Figs 11, 12, 62). Chaetotaxy well developed, setae capilliform, variable in length, greyish or yellowish. Each side of prothorax (Fig. 59) with 11 prns of almost equal size (8 of them placed on the weakly visible premental sclerite, next 3 close to the spiracle; 2 ps moderately long and 1 short eus. Meso- and metathorax on each side with 1 moderately long prs and 4 pds of various lengths (first, second and fourth very short, third long), 1 moderately long and 1 short as, 1 moderately long and 1 short ss, 1 eps and 1 ps, both moderately long, 1 short eus. Each pedal area of thoracic segments with 6 pda, almost equal in length. Abd. 1-7 (Figs 60-63) on each side with 1 moderately long prs and 5 pds, of various lengths (first, second and fourth very short, third and fifth very long; Abd. 7 with 6 pds), arranged along the posterior margin of each segment, 1 long ss, 2 eps and 2 ps, both of different lengths, 1 lsts and 2 moderately long eus. Abd. 8 (Figs 61-63) on each side with 1 moderately long prs, 4 relatively elongate pds, 2 eps and 2 ps, both of different lengths, 1 lsts and 2 eus. Abd. 9 (Figs 61-63) on each side with 3 ds, first and third moderately long, second short, all located close to the posterior margin of the segment, 1 medium ps and 2 short sts. Each lateral anal lobe (Abd. 10) with 3 short setae (ts1-3). Figures 59-63. Tanymecus palliatus mature larva, habitus and chaetotaxy. 59 Thoracic segments lateral view 60 First abdominal segment lateral view 61 Abdominal segments 7-10 lateral view 62 Abdominal segments 7-10 ventral view 63 Abdominal segments 7-10 dorsal view. Abbreviations: Th. 1-3 - thoracic segments 1-3, Abd. 1-10 - abdominal segments 1-10, setae: as - alar, ps - pleural, eps - epipleural, ds - dorsal, lsts - laterosternal, eus - eusternal, pda - pedal, pds - postdorsal, prns - pronotal, prs - prodorsal, sps - spiracular, sts - sternal, ts - terminal. Head (Fig. 64). Greyish or light yellowish, suboval, frontal suture distinct, Y-shaped, endocarina absent. Setae on head capilliform; des1, 2, 3, 5 equal in length; des1 and des2 located in the central part of epicranium, des3 placed on frontal suture, des5 located anterolaterally; fs4, 5 both as long asdes1, fs4 located anteromedially, fs5 anterolaterally, near epistome; les1 and les2, equal in length, only slightly shorter than des1; ves1 and ves almost as long asles. Postepicranial area with 5 very short pes (Fig. 64). Antenna (Fig. 65) located at the end of frontal suture; antennal segment membranous, bearing cushion-like sensorium (Se), located medially, and 6 sensilla of various types: 5 ampullacea (sa) and 1 basiconicum (sb). Labrum (Fig. 66) narrow, anterior margin slightly sinuate; 3 pairs of lrs, lrs1 and lrs3 moderately long, lrs2 long; lrs1 anteromedially, lrs2 medially, lrs3 laterally. Clypeus (Fig. 66) twice as long as than labrum, anterior margin of clypeus straight; 2 pairs of cls: cls1as long aslrs2, cls2 less than half the length of cls1, both located posteromedially; clss clearly visible, placed medially between cls. Epipharynx (Fig. 66) with 4 pairs of finger-shaped als of equal length; 3 pairs of ams: ams1 rod-shaped, very short, ams2 and ams3 moderately long, finger-like; 2 pairs of short, rod-shaped mes, both placed medially, between labral rods. Surface of epipharynx smooth. Labral rods short, converging posteriorly. Mandibles (Fig. 67) curved, narrow, with slightly divided apex (teeth of various length). There is an additional tooth on the cutting edge in the middle of the mandible; both mds capilliform, different in length. Maxilla (Figs 68-70) with 1 stps and 2 pfs of equal length; mala with 7 rod-like dms of various size (Fig. 69) and 4 capilliform vms variable in length(Fig. 70); mbs short. Maxillary palpi with 2 palpomeres, basal with short mps; distal palpomere apically with a group of sensilla, each palpomere with a pore. Basal palpomere distinctly wider and slightly longer than distal. Prelabium (Fig. 68) almost rounded with 1 long prms, located medially. Ligula with 2 pairs of ligs of various length. Premental sclerite clearly visible tri den -shaped, median branch and posterior extension weakly sclerotized. Labial palpi 2-segmented; apex of distal palpomere with some sensilla; each palpomere with a pore. Basal palpomere wider and longer than distal. Postlabium (Fig. 68) with 3 capilliform pms, the first pair located anteromedially, the remaining 2 pairs posterolaterally; pms1 and pms3 long, pms2 very long. Only posterior margin of postlabium covered with fine asperities. Figure 64. Tanymecus palliatus mature larva, head, frontal view. Abbreviations: at - antenna, setae: des - dorsal epicranial, fs - frontal, les - lateral epicranial, pes - postepicranial, ves - ventral. Figure 65. Tanymecus palliatus mature larva, body parts, right antenna. Abbreviations: Se - sensorium, sa - sensillum ampullaceum, sb - sensillum basiconicum. Figure 66. Tanymecus palliatus mature larva, body parts, clypeus, labrum and epipharynx. Abbreviations: clss - clypeal sensorium, lr - labral rods, setae: als - anterolateral, ams - anteromedial, cls - clypeal, lrs - labral, mes - median. Figure 67. Tanymecus palliatus mature larva, left mandible. Abbreviations: mds - mandibular seta. Figures 68-70. Tanymecus palliatus mature larva, body parts. 68 Maxillolabial complex ventral aspect 69 Right maxilla apical part dorsal aspect 70 Right maxilla apical part ventral aspect. Abbreviations: setae: dms - dorsal malar, ligs - ligular, mbs - malar basiventral, mps - maxillary palp, pfs - palpiferal, prms - prelabial, pms - postlabial, stps - stipal, vms - ventral malar. Differentiation of described species

Emden 1950	2
Emden 1950	3
dspdassSe	Graptus triguttatus triguttatus
dspdassSe	Peritelus sphaeroides
dspdassepsasas	Philopedon plagiatum
dspdassepsasas	Tanymecus palliatus

The number of larval instars in Tanymecus There are some strange statements about the number of larval instars in the larval stage of species of genus Tanymecus . Hoffmann (1963) , who relied on authors from the former Soviet Union, reported about 10 larval instars in T. palliatus , which was already commented by Dieckmann (1983) as a 'for weevils surprising fact' . In T. dilaticollis Gyllenhal, 1834, Catrinici (1944) determined six larval instars. She reported that larval head width increased up to the fourth larval instar, decreased in the fifth and increased again in the sixth instar to nearly the same value as in the fourth. This sounds really strange and has to be taken with caution and tested with new observations. This was also the reason for Van Emden (1952) to propose four larval instars for T. dilaticollis . For the exact determination of the number of larval instars we summarized and assessed our own measuring data and added data from literature, if necessary (Tables 1, 2). Due to the dubiousness of the number of larval instars in T. dilaticollis given by Catrinici (1944) and Van Emden (1952) we used measuring data for the head width (HW) of adults of both species and of mature larvae of T. palliatus to assess the HW of the mature larva of T. dilaticollis . This ratio should be rather similar in two species of the same genus. Hence, the value calculated in this way for the HW of the mature larva of T. dilaticollis is 1.51 mm. We also needed to determine the number of larval instars for both species: there are data for L1 and for mature larvae, and in T. dilaticollis there are also measurements for several instars, even if (especially in the higher instars) the data are doubtful. The determination of larval instars is mainly based on the method of Dyar (1890) and has been used by several authors, even if apparently not known to all scientists who have dealt with larvae. There are several publications about weevil larvae where this ratio was applied. We preferred to use Dyar's ratio- 1 and called it Growth Factor (GF) as it corresponds more to the natural development. In Mitoplinthus caliginosus (Fabricius, 1775) (subfamily Molytinae ), after comparison of the growth factors 1.35, 1.4 and 1.5, the best approximation was found with a value of 1.4 for head capsule width ( Sprick and Gosik 2014 ). This value agrees with Dyar's ratio of 0.714. Rowe and Kok (1985) gave a Dyar's ratio value for Rhinocyllus conicus ( Froelich , 1792) (subfamily Lixinae ) larvae of 0.65 (this agrees with a GF of 1.538). In agreement Leibee et al. (1980) determined the ratios of each instar of two populations of Sitona hispidulus (Fabricius, 1777) (subfamily Entiminae , tribe Sitonini ) and reported Dyar's values between 0.642 and 0.739. The median value of these data is by our calculation 0.6995 (GF = 1.43). These data show that there are rather different values for larval growth and that there are also differences between the growth of different larval instars. For larval instar determination in Tanymecus we tested four values between 1.4 and 1.5 to achieve the best approximation of larval growth. We started with the L1 larva that we received from egg-laying of adult weevils (head width 0.38 mm) and calculated the subsequent instars with the selected GF values until 1.71 mm, the head width of the mature larvae, was achieved. For this procedure, five steps were needed. Higher GF values, as for example 1.538 in Rhinocyllus conicus , were excluded because of the reduced number of larval instars in this rather distantly related subfamily (Table 3). From Table 2 it can easily be seen that both species have 5 larval instars. The best approximation is achieved with a GF of 1.44 in Tanymecus dilaticollis and 1.46 in T. palliatus (i.e. Dyar's ratio of 0.694 and 0.685, respectively). The small difference may be due to the absent HW variation of the two available adult T. dilaticollis specimens that showed both the same value and hence do not represent the HW variation of the population. Furthermore it can be stated that the values of Catrinici (1944) are beginning to seem doubtful from the fourth larval instar onward. For this approximation it is only necessary to know the head width of the L1 larva and that of the last instar. And the HW of the last instar can be assessed from the HW of the adult weevil, as it is shown in Table 2. In adults HW was always measured directly behind the eyes to avoid an excessive importance of prominent eyes, which could be a problem in genera such as Strophosoma Billberg, 1817 (see Gosik et al. 2017 ) or in species such as Tanymecus dilaticollis . Larval growth, number of larval instars and size of the adults' head width (and therefore size of adults, too) are in a very close relationship to each other. The same may be true for the HW of the pupa. An instar determination is also possible for Graptus triguttatus . According to Van Emden (1952) , the head capsule width of the L1 larva is 0.34 mm (average of three larvae; Table 1). The application of a GF of 1.45 shows a good approximation with the measuring values given in Table 1: 0.34 mm x 1.45 (repeatedly) = 0.493 mm (L2), 0.715 mm (L3), 1.037 mm (L4) and finally 1.503 mm (L5). Thus, Graptus triguttatus has also 5 larval instars, and the premature larvae from Table 1 may represent L2 and L4 larvae. There is a great variation in adults' head width in this species ranging from 1.05 mm to 1.5 mm (Table 1). This agrees with the span between the last larval instars and nearly achieves the supposed growth factor of 1.45, so that the assignment of a certain larva to the right instar is doubtful in extremely sized specimens, the more as head width variation becomes larger with instar and size. A similar size variation was observed in Peritelus sphaeroides adults and Philopedon plagiatum larvae (Table 1). In Peritelus sphaeroides and Philopedon plagiatum an instar determination is impossible due to the absence of L1 head width data. It can only be concluded from the data for premature larvae in Philopedon plagiatum (Table 1) that these data represent the penultimate instar. Opposite to Graptus and Tanymecus , the HW of Philopedon adults is greater than in mature larvae. In Peritelus sphaeroides the HW of adults is slightly smaller than in mature larvae, but not significantly. Table 1. Head width measuring data of the species studied. Results in mm; n - number of specimens measured, in adults behind eyes; L1 - first instar larva; ML - mature larva; *: an assignment to this instar is doubtful. A transfer to 'mature larvae' would change the average value only slightly; **: data from Gosik and Sprick (2013) . Data from literature in italics.

Species	Larval instars			Pupa	Adult
	L 1 larvae	Premature larvae	Mature larvae
Graptus triguttatus 1 Van Emden 1952	0.3310,3410.351	23121	3212641	111	11133111
Peritelus sphaeroides	12534	11	112211
Philopedon plagiatum	141	111	121112
Philopedon plagiatum Van Emden 1952	0.572; 0.611; 0.631; 0.641; 0.681; 0.721; 0.751*; 0.791	0.861; 0.892; 0.962; 1.001; 1.072; 1.081; 1.12; 1.141; 1.171; 1.181; 1.212
Tanymecus dilaticollis	2
Tanymecus palliatus	12522	111	11111

Table 2. Head width measuring data of Tanymecus larvae and adults for larval instar determination. *: collected together with T. palliatus pupae; **: calculated from the ratio of adult's HW to HW of the mature larva.

Instar	Mean value (mm)	Specimens measured	Source
Tanymecus dilaticollis
1	Catrinici (1944)
2	Catrinici (1944)
3	Catrinici (1944)
4	Catrinici (1944)
1.51
Tanymecus palliatus
1	1
1
Van Emden (1952)

Table 3. Larval instar determination for Tanymecus dilaticollis and T. palliatus . All measuring data in mm; initial data bold, calculated data in italics; target data of the approximation bold and in italics.

Tanymecus palliatus
1	0.38	0.38	0.38
2	0.532	0.551/0.555	0.57
3	0.745	0.799/0.810	0.855
4	1.042	1.158/1.183	1.283
5	1.460	1.680/1.727	1.924
1.71	1.71	1.71
Tanymecus dilaticollis
1	0.35	0.35	0.35
2
2	0.49	0.504/0.508/0.511	0.525
3
3	0.686	0.726/0.736/0.746	0.7875
4
4	0.960	1.045/1.067/1.089	1.181
5	1.345	1.505/1.55/1.590	1.772
1	1.51	1.51	1.51

Table 4. Number of setae in mature larvae of Graptus triguttatus triguttatus (a), Peritelus sphaeroides (b), Philopedon plagiatum (c), Tanymecus palliatus (d).

Part of body	Setae	a	b	c	d
prns
ps
eus
pda
prs
pds
as
ss
eps
ps
eus
pda
prs
prs
pds
pds
ss
ss
eps
eps
ps
ps
lsts
lsts
eus
eus
ds
ps
sts
ts
des
fs
ls
pes
ves
cls
lrs
mes
als
ams
mds
des
ves
pfs
stps
pbs
mps
pms
prms
ligs