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Figure 12 in Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Figure 12. Hansenocaris demodex sp. nov., early nauplius (LSN − 5 or LSN − 6?) from Sesoko Island in SEM. A, ventrolateral view; B, lateral view; C, dorsal view; D, frontal view; E, labrum and naupliar appendages, right side; F, naupliar appendages, right side, median view; G, first and second antennae, right side, lateral view, asterisk indicating rudimentary segment of a2 exopod; H, dorsocaudal spine and furcal spines, terminal view. Abbreviations: a1, first antenna; a2, second antenna; ba, basis; co, coxa; dc sp, dorsocaudal spine; en, endopod; ex, exopod; fur sp, furcal spine; F-1 to F-3, frontal plates 1–3; la, labrum complex; md, mandible; rud, rudimentary; W, window plate. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to those cuticular structures that could be matched with those of the LSN (see Table 4). Yellow overlay, cephalic shield; blue overlay, faciotrunk; red overlay, anterior field of facets. Square with dotted outline: museum number.

Figure 10 in Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Figure 10. Hansenocaris demodex sp. nov., cyprid from Green Island in SEM. A, lateral view; B, cephalic shield, frontal view; C, putative lattice organ, close-up; D, thoracic limbs 1–6; E, thorax, abdomen and telson, dorsal view. Abbreviations: ba, basis; co, coxa; en, endopod; ex, exopod; la, labrum; pro scle, proximal sclerites of thoracopods; thp 1, thoracopod 1; thp 6, thoracopod 6. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 5). Square with dotted outline: sample number and museum numbers. Dotted arrows in D point to long medial setae of thoracopodal endopods.

Figure 9 in Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Figure 9. Hansenocaris demodex sp. nov., holotype (A, B, D, E) and paratype (C) cyprid from Sesoko Island in SEM. A, posterior thoracic segments and anterior abdominal segments, lateral view; B, thoracopods, ventral view; C, telson, dorsall view; D, telson, terminal view; E, right furcal ramus, posterior view. Abbreviations: fur ram, furcal ramus; ba, basis; co, coxa; en, endopod; ex, exopod; pro scle, proximal sclerites of thoracopods; thp 1, thoracopod 1. Roman numerals I–VI – thoracic segments. Large Arabic numerals 1–3 – abdominal segments 1–3. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 5). Square with dotted outlines: sample numbers, museum numbers and type status.

Figure 14 in Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Figure 14. Morphology of four types of y-larvae from Sesoko Island used for molecular analyses (see overview in Table 6), all photographed in life. A, Hansenocaris demodex sp. nov., lecithotrophic LSN (last-stage nauplius) with cyprid inside. B, 'Bumblebee' type, lecithotrophic LSN with cyprid inside. C, 'Big brown' type, lecithotrophic LSN with cyprid inside. D, planktotrophic nauplius, identified as Itô's (1986a) Pacific type I. Squares with dotted outline: sample numbers.

Figure 6 in Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Figure 6. Hansenocaris demodex sp. nov., last-stage nauplius (LSN) from Green Island in SEM. A, lateral view; B, dorsal view. C, ventral view; D, frontal view. Abbreviations: a1, first antenna; a2, second antenna; dc sp, dorsocaudal spine; fur sp, furcal spine; la, labrum; md, mandible. Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 4). Square with dotted outline: sample number and museum number.

Figure 8 in Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Figure 8. Hansenocaris demodex sp. nov., holotype (B, C) and paratypes (A, D), cyprids from Sesoko Island in SEM. A, live specimen in four different positions; B, cephalic shield, frontal view; C, cephalic shield, dorsal view; D, cephalic shield, oblique posterior view. Abbreviations: par occ pro, paraocular process. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures of the cyprid (see Table 5). Squares with dotted outlines: sample numbers, museum numbers and type status.

Figure 7 in Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Figure 7. Hansenocaris demodex sp. nov., holotype (A, B, D–I) and paratype (C), cyprids from Sesoko Island photographed before drying (A) and in SEM after drying and mounting (B–I). A, lateral view; B, lateral view; C, cephalic region, lateral view; D, cephalic region, ventral view; E, bifid appendix (frontal filaments?); F, labrum; G, pores and sensilla; H, close-up of hook of left first antenna; I, abdomen and telson, dorsal view. Abbreviations: a1, first antenna; a1 hk, first antenna hook; a2 rud, second antenna rudiment; ae, aesthetasc; en, endopod; ex, exopod; la, labrum; md rud, mandible rudiment; par occ pro, paraocular process. Roman numerals I–VI – thoracic segments. Large Arabic numerals 1–3 – abdominal segments. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 5). Square with dotted outline: sample numbers, museum numbers and type status.

Figure 5 in Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Figure 5. Hansenocaris demodex sp. nov., paratype, last-stage nauplius (LSN) from Sesoko Island in SEM. A, lateral view; B, cephalic shield, anterior view; C–J, close-up views of pores and sensilla of cephalic shield and faciotrunk; K–M, labrum and naupliar limbs (a1, a2, md). Abbreviations: a1, first antenna; a2, second antenna; md, mandible; la, labrum; scl, sclerite of a1. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 4). Yellow overlay, cephalic shield; blue overlay, faciotrunk; red overlay, anterior field of facets. Square with dotted outline: sample number, museum number and type status.

Figure 4 in Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Figure 4. Hansenocaris demodex sp. nov., paratype, last-stage nauplius (LSN) from Sesoko Island photographed live before fixation (A) and in SEM after fixation and mounting (B–H). A, ventral view; B, ventral view; C, dorsal view; D, pair of ventral pores situated in front of labrum; E, close-up views of pores and sensilla of cephalic shield; F, caudal end in dorsal view; G, caudal end in ventral view; H, caudal end viewed from behind. Abbreviations: a1, first antenna; a2, second antenna; arthro membr, arthrodial membrane; ce, compound eye; dc sp, dorsocaudal spine; en, endopod; ex, exopod; fur sp, furcal spine; la, labrum; md, mandible; ne, nauplius eye; rud, rudimentary. Small Arabic numerals, many annotated with l or r for left and right, respectively, refer to cuticular structures (see Table 4). Yellow overlay, cephalic shield; blue overlay, faciotrunk; red overlay, anterior field of facets. Square with dotted outline: sample number, museum number and type status.

Figure 3 in Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Figure 3. Overview of most specimens of Hansenocaris demodex sp. nov. collected at Sesoko Island during fieldwork in 2018 and 2019. A, 19 specimens, four of which reached the cyprid stage, collected in the plankton at the naupliar stage and reared individually, with length, dish number, sample number, and museum registration number indicated for each specimen as well as a colour code (red, blue, yellow) showing its ultimate treatment/fixation (respectively, molecular work, SEM or exuvium on slide); some larvae died before preservation and are only referred to by their dish number; B, overview of setup for individual rearing of larvae; top view of green tray containing 31 2.5-cm-wide dishes with tracking information written on lids, each containing one to five live larvae. Live video of H. demodex and other y-larvae can be seen here: https:// youtu.be/seo-63AK10E.

Figure 1 in Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Figure 1. Facetotecta sampling sites in East Asia, 2017–20 and distribution of Hansenocaris demodex sp. nov. A, East China Sea showing distance (780 km) between the two main sampling sites, Sesoko Island (Okinawa, Japan) and Green Island (Taiwan). In 2020, several additional specimens of H. demodex were collected from Xiaoliuqiu Island (MJG, unpublished). B, Sesoko Island (Japan, Okinawa) with indication of sampling site (Sesoko Station); C, Green Island (Taiwan) with indication of sampling site (Gonguan Fishing Harbour); D, pier at Sesoko Station; E, Gonguan Fishing Harbour.

Figure 2 in Integrative taxonomy of crustacean y-larvae (Thecostraca: Facetotecta) using laboratory-rearing and molecular analyses of single specimens, with the description of a new vermiform species

Figure 2. Example of newly collected sample of still living y-larvae collected on 19 October 2018 at Sesoko Island off the pier shown in Figure 1D, representing a variety of developmental stages. A, 25 y-larvae likely representing> 15 different species, including 18 early lecithotrophic nauplii (*), five planktotrophic nauplii (†) and two cyprids (§). None of the depicted specimens can be assigned to already described species, but three of the planktotrophic nauplii are similar to Itô's (1986a) Pacific type I and one is a cyprid of the type nicknamed 'Big brown', both types of which are sequenced as a part of this work and belong to the same clade as Hansenocaris demodex sp. nov. (see Fig. 15). B, close-up of selected y-larvae with same numbers assigned as in A. Live video of H. demodex and other y-larvae can be seen here: https://youtu.be/seo-63AK10E.

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Immune assay data for Lycaeides melissa larvae reared on different host plants and with/without egg microbes

1. Maternally transmitted microbes are ubiquitous. In insects, maternal microbes can play a role in mediating the insect immune response. Less is known about how ecological factors, such as resource use, interact with maternal microbes to affect immunity. 2. In the context of a recent colonization of a novel host plant by the Melissa blue butterfly (Lycaeides melissa), we investigated the interaction between host plant use and vertically transmitted, extracellular egg-associated microbes in determining the strength of the insect immune response. 3. We reared larvae on two different host plant species: a native host Astragalus canadensis and a novel host Medicago sativa. Egg-associated microbes were removed through a series of antimicrobial egg washes prior to hatching. Immune response was measured through three assays: standing phenoloxidase (PO), total PO, and melanization. 4. We detected strong effects of microbial removal. Egg washing resulted in larvae with an increased immune response as measured by total PO- contrary to reports from other taxa. The effect of washing was especially strong for larvae consuming the native host plant. 5. This result may explain why consumption of the egg casing is not a universal behaviour in insects, due to negative effects on larval immunity.

FIGURE 7 in Larval development of the crab Amphithrax hemphilli (Rathbun, 1892) (Decapoda, Brachyura, Mithracidae) described from laboratory-reared material

FIGURE 7. Cladogram of species of Mithrax and allies for the megalopa stage, with phylogeny constructed from Windsor & Felder (2014) and Assugeni et al. (2017). Characteristics: (1) setae on the peduncle of the antennule; (2) aesthetascs on the exopod of the antennule; (3) setation of the antenna; (4) setae on the palp of the mandible; (5) setae on the coxal endite of the maxillule; (6) setae on the basial endite of the maxillule; (7) setae on the endopod of the maxillule; (8) setae on the epidod of the maxillule; (9) setae on the coxal endite of the maxilla; (10) setae on the basial endite of the maxilla; (11) setae on the endopod of the maxilla; (12) setae on the scaphognathite of the maxilla; (13) setae on the basis of the first maxilliped; (14) setae on the endopod of the first maxilliped; (15) setae on the exopod of the first maxilliped; (16) setae on the endopod of the second maxilliped; (17) setae on the exopod of the second maxilliped; (18) setae on the coxa of the third maxilliped; (19) setae on the basischium of the third maxilliped; (20) setae on the endopod of the third maxilliped; (21) setae on the exopod of the third maxilliped, and (22) setae on the pleon. Different colors indicate differences among species; equal colors indicate similarity.

FIGURE 4 in Larval development of the crab Amphithrax hemphilli (Rathbun, 1892) (Decapoda, Brachyura, Mithracidae) described from laboratory-reared material

FIGURE 4. Amphithrax hemphilli (Rathbun, 1892), megalopa stage. (A) maxilliped I; (B) maxilliped II; (C) maxilliped III; (D) pleopods 1–4 and uropod; (E) pereiopods 1–5; (F) dorsal and lateral view of pleon and telson. Scale bars: A–C: 0.1 mm; D–F: 0.2 mm.

FIGURE 1 in Larval development of the crab Amphithrax hemphilli (Rathbun, 1892) (Decapoda, Brachyura, Mithracidae) described from laboratory-reared material

FIGURE 1. Amphithrax hemphilli (Rathbun, 1892), first zoeal stage. (A) Lateral view; (B) dorsal view; (C) antennule; (D) antenna; (E) mandible; (F) maxillule (G) maxilla; (H) maxilliped I; (I) maxilliped II; (J) maxilliped III; (K) pereiopods; (L) dorsal view of pleon and telson. Scale bars: A, B, L: 0.2 mm; C–K: 0.1 mm.

FIGURE 6 in Larval development of the crab Amphithrax hemphilli (Rathbun, 1892) (Decapoda, Brachyura, Mithracidae) described from laboratory-reared material

FIGURE 6. Cladogram of species of Mithrax and allies for the second zoeal stage, with phylogeny constructed from Windsor & Felder (2014) and Assugeni et al. (2017). Characteristics: (1) setae on the anterodorsal region of the carapace; (2) setae on the posterodorsal region of the carapace; (3) setae on the ventral margin of the carapace; (4) aesthetascs on the antennule; (5) setae on the antennule; (6) setae on the coxal endite of the maxillule; (7) setae on the basial endite of the maxillule; (8) setae on the endopod of the maxillule; (9) setae on the coxal endite of the maxilla; (10) setae on the basial endite of the maxilla; (11) setae on the endopod of the maxilla; (12) setae on the scaphognathite of the maxilla; (13) setae on the coxa of the first maxilliped; (14) setae on the basis of the first maxilliped; (15) setae on the endopod of the first maxilliped; (16) setae on the coxa of the second maxilliped; (17) setae on the basis of the second maxilliped, and (18) setae on the endopod of the second maxilliped. Different colors indicate differences among species; equal colors indicate similarity.

FIGURE 2 in Larval development of the crab Amphithrax hemphilli (Rathbun, 1892) (Decapoda, Brachyura, Mithracidae) described from laboratory-reared material

FIGURE 2. Amphithrax hemphilli (Rathbun, 1892), second zoeal stage. (A) Lateral view; (B) antennule; (C) antenna; (D) mandible; (E) maxillule (F) maxilla; (G) maxilliped I; (H) maxilliped II; (I) maxilliped III; (J) pereiopods; (K) dorsal view of pleon and telson. Scale bars: A, J, K: 0.2 mm; B–I: 0.1 mm.

FIGURE 5 in Larval development of the crab Amphithrax hemphilli (Rathbun, 1892) (Decapoda, Brachyura, Mithracidae) described from laboratory-reared material

FIGURE 5. Cladogram of species of Mithrax and allies for the first zoeal stage, with phylogeny constructed from Windsor & Felder (2014) and Assugeni et al. (2017). Characteristics: (1) setae on the anterodorsal region of the carapace; (2) setae on the posterodorsal region of the carapace; (3) setae on the ventral margin of the carapace; (4) aesthetascs on the antennule; (5) setae on the antennule; (6) setae on the coxal endite of the maxillule; (7) setae on the basial endite of the maxillule; (8) setae on the endopod of the maxillule; (9) setae on the coxal endite of the maxilla; (10) setae on the basial endite of the maxilla; (11) setae on the endopod of the maxilla; (12) setae on the scaphognathite of the maxilla; (13) setae on the coxa of the first maxilliped; (14) setae on the basis of the first maxilliped; (15) setae on the endopod of the first maxilliped; (16) setae on the coxa of the second maxilliped; (17) setae on the basis of the second maxilliped, and (18) setae on the endopod of the second maxilliped. Different colors indicate differences among species; equal colors indicate similarity.